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Autism spectrum disorder and pupillometry: A systematic review and

meta-analysis

Lyssa de Vries, Iris Fouquaet, Bart Boets, Gunnar Naulaers, Jean

Steyaert

PII: S0149-7634(20)30590-X
DOI: https://doi.org/10.1016/j.neubiorev.2020.09.032
Reference: NBR 3924

To appear in: Neuroscience and Biobehavioral Reviews

Received Date: 30 March 2020

Revised Date: 31 July 2020
Accepted Date: 24 September 2020

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Autism spectrum disorder and pupillometry: a systematic
review and meta-analysis
Lyssa de Vries *a,b,c, Iris Fouquaet a, Bart Boets a,c, Gunnar Naulaers d, Jean Steyaert a,b,c

* Corresponding author: lyssa.devries@kuleuven.be,

Kapucijnenvoer 7 - block h postbus 7003, 3000 Leuven, Belgium

Affiliations:

a. Center for Developmental Psychiatry, Department of Neurosciences, KU Leuven, Kapucijnenvoer 7,

3000 Leuven, Belgium.

b. Department of Child Psychiatry, UPC KU Leuven, Herestraat 49, 3000 Leuven, Belgium.

c. KU Leuven Autism Research (LAuRes), KU Leuven, Leuven, Belgium

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d. Department of Development and Regeneration, University Hospitals Leuven, Neonatal Intensive
Care Unit, Herestraat 49, 3000 Leuven, Belgium

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Highlights



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Pupillometry measures how a pupil reacts to a light flash or a more complex stimulus
The pupil is a window into the autonomic nervous system
 Latency of the pupil response is longer in individuals with autism spectrum disorder
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 Explanations for underlying processes vary widely in current literature
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Abstract
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Pupillometry, measuring pupil size and reactivity, has been proposed as a measure of autonomic
nervous system functioning, the latter which might be altered in individuals with autism spectrum
disorder (ASD). This study aims to evaluate if pupillary responses differ in individuals with and
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without ASD. After performing a systematic literature search, we conducted a meta-analysis and
constructed a qualitative synthesis. The meta-analysis shows a longer latency of the pupil response in
the ASD-group as a substantial group difference, with a Hedges’ g of 1.03 (95%CI 0.49-1.56, p=0.008).
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Evidence on baseline pupil size and amplitude change is conflicting. We used the framework method
to perform a qualitative evaluation of these differences. Explanations for the group differences vary
between studies and are inconclusive, but many authors point to involvement of the autonomous
nervous system and more specifically the locus coeruleus-norepinephrine system. Pupillometry
reveals differences between people with and without ASD, but the exact meaning of these
differences remains unknown. Future studies should align research designs and investigate a possible
effect of maturation.

Keywords: Autism spectrum disorder, pupillometry, eye-tracking, meta-analysis, qualitative

research, autonomic nervous system
Introduction
New technologies, such as pupillometry, have gained increasing interest and provide insight into the
autonomic nervous system (ANS) functioning of an individual. The pupil is considered as a window
into the ANS, as an index of the sympathetic and the parasympathetic nervous system functioning 1.
The sympathetic nervous system is responsible for the fight/flight reactions of the body to stress,
whereas the parasympathetic nervous system is involved in the freeze response to stress and
regulating the vital bodily functions in rest.

ANS functioning can be measured in many ways besides through pupillometry, for example by heart
rate variability (HRV) or skin conductance response (SCR).2 The pupillary response is quick and easy
to record. Cardiac measurements of the ANS, such as heart rate variability or respiratory sinus
arrhythmia, are also reliable, yet have several limitations that pupillometry doesn’t: they are
influenced by the level of habitual physical activity, age or health of the participants and by

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differences in breathing protocols or analytical differences such as using different bandwidths. Many
different parameters are used in cardiac measurement of the ANS, which makes it hard to compare
different studies3. In comparison with other measurements of the ANS, pupillometry is even less

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invasive, yet fast and easy to perform, thus ideally suited for testing ANS in paediatric and clinical
populations such as individuals with autism spectrum disorder (ASD).
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ASD is a neurodevelopmental condition, defined by socio-communicative difficulties and repetitive
and restrictive behaviours or interests4. The understanding of the aetiology and underlying
mechanisms is still growing. Genetic predisposition and environmental factors play a role5. Diagnosis
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is difficult because individuals with ASD present with a wide diversity of phenotypes.

Previous research has suggested ANS dysfunction in ASD. According to the Polyvagal Theory of
Porges6 , the ANS has been linked to communication, a key deficit domain in the ASD phenotype. This
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theory states that from an evolutionary point of view, development of the ANS was a crucial survival
strategy in mammals as it perfected adaptive behaviours, amongst which communication. According
to this theory, a certain physiologic state limits the range of behaviours. The theory proposes three
phylogenetic subsystems linked to social communication, mobilization and immobilization. A soothed
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autonomic state is proposed to be associated with social abilities, but when the environment is not
perceived as safe and arousal increases, social behaviours are compromised7,8. It has long been
suggested that some individuals with ASD have a hyperaroused ANS9. However, many other
researchers have disputed this10. Still others speculate that acetylcholine, a neurotransmitter in the
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ANS, is dysregulated in individuals with ASD11.

Pupillary response
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Pupillometry refers to the measurement of changes in pupil size over time, often in response to a
visual stimulus. It has been used in participants of all ages, from infants to adults, allowing the study
of developmental aspects12,13. Pupillometry is currently used in the context of both physiological and
psychological responses14. First, the most basic pupillary light response (PLR) is a dynamic reflexive
change of the pupil size as a response mediated by the brain to a simple luminance change detected
on the retina. Alternatively, in psychological research, the pupillary response to more complex and
meaningful visual stimuli (which we will further refer to as PRC) has been shown to be an index of
either an orienting response or of mental effort, cognitive load or more specific cognitive processes
such as emotional processing15. Accordingly, this pupillary response to more complex and meaningful
visual stimuli reflects the increase of physiological arousal associated with sympathetic activation.
The pupillary reaction is most often recorded with a commercially available or self-constructed eye-
tracker, but in older studies video recordings have been used. Eye-trackers can provide detailed data
on the dilation and constriction amplitude, as well as on the time frame in which the pupil reacts, to
obtain a full view of the pupillary response waveform.

The pupillary response to light is mediated by various structures in the brain (Figure 1). Light passes
through the pupil and falls on the retina. The afferent pathway continues when, by stimulation of the
photosensitive retinal ganglion cells, the photoreceptors (rods and cones) are activated. The signal is
transferred through the optic nerve (CN-II) and through the optic chiasm, towards the pretectal
nuclei. From these midbrain nuclei, the signal passes towards another close by nucleus: the nucleus
of Edinger-Westphal, ipsilaterally and contralaterally. Starting there, the preganglionic
parasympathetic fibres travel along the oculomotor nerve (CN-III), synapsing in the ciliary ganglion
with the post-ganglionic fibres before returning back to the iris, where they innervate the contracting
sphincter muscle, which causes miosis.1

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Besides this parasympathetically mediated pathway, the sympathetic system also mediates pupil
dilation through innervation of the pupil dilator muscle. The sympathetic afferent pathway arises
from thoracic and cervical ganglia, where several sympathetic motor neurons are located, and

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continues by the carotid plexus into the ciliary nerve to the pupil dilator muscle, causing mydriasis.
The eye is under further autonomic control by several afferent nerves, both parasympathetic and
sympathetic, coming from the superior salivatory nucleus, the pterygopalatine ganglion, the
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intermediolateral cell column, the superior cervical ganglion and the trigeminal nerve, amongst
others.2,16 Being both parasympathetic and sympathetic innervated, the pupil shows direct feedback
of the ANS when responding to stimuli. Higher-order brain regions outside of these pathways, such
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as the prefrontal cortex, can influence both systems by effecting upon medullar and midbrain
nuclei.17.

Multiple authors pinpoint the locus coeruleus norepinephrine (LC-NE) system as a key underlying
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mechanism involved in the pupillary response18,19. The LC is the source of norepinephrine in the brain
and is thus involved in arousal14. It is part of the sympathetic nervous system and has an important
role in attention19. Animal studies have shown that pupil dilation is phase locked with NE-release in
the LC20. Aston-Jones & Cohen propose the adaptive-gain theory, where two modes of activity are
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defined. On the one hand, phasic activity of the LC, associated with intermediate or small pupil size,
is related to exploitation, engaging in one single task and optimizing this task performance. Tonic
activity on the other hand, resulting in pupil dilation, is found during exploration, switching quickly
between tasks 15,20.
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In relation to the LC-NE system, different attention capacities have been suggested as an explanation
for differences in pupil response between ASD and TD population. Alerting, attention orienting and
executive functioning are three systems that are under control of the LC-NE system19.
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Furthermore, Lynch hypothesizes that a disruption of cranial nerve nuclei could explain a deviant
pupillary response in ASD, based on a previously described model of underconnectivity between the
prefrontal cortex and other brain regions in ASD on the one hand and a valproic acid animal model
on the other hand, which both show the importance of cranial nerve nuclei and their connections to
other brain regions.18

Present study
In this study, we focus on the three most commonly measured pupil characteristics: baseline pupil
size, latency and amplitude change of constriction/dilation. The classical pupillary waveform in
response to light (i.e. PLR) is shown in Figure 2. In response to other more complex stimuli, the
expected waveform will differ according to the corresponding research design.

The aim of this review is to summarise the available evidence on the characteristics of the pupillary
response in individuals with ASD as compared to typically developing individuals. We include both
empirical data and theoretical explanatory models in a qualitative analysis. Furthermore, we
analysed quantitative differences and performed a meta-analysis.

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Methods
Literature search
We conducted a systematic search of multiple databases (Pubmed, Embase, Web of Science) using
combinations of the search terms ‘autism’, ‘autism spectrum disorder’, ‘ASD’, ‘pupil’, ‘pupil reflex’,
‘pupillary light reflex’. . The PRISMA checklist was used during the different stages of this review.21
Databases were searched for published articles from the start of the database until June 2020
Articles were screened, based on title and abstract, to evaluate if they fulfilled the following inclusion
criteria: (1) experimental design, (2) human population, (3) comparison between ASD and a control
group, (4) English language. Afterwards, we evaluated the full text.

Risk of bias
Articles were assessed for the risk of bias using the QUADAS-2 tool22, which is designed to assess the
quality of diagnostic accuracy studies. Studies were evaluated at four levels: patient selection, index

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test, reference standard and flow/timing. The method of patient selection was evaluated, for
example if this was a random or a consecutive sample of patients enrolled. The index test,
pupillometry, should be described in detail in order to evaluate its conduct and interpretation, e.g.

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were the index test results interpreted without knowledge of the reference standard. The reference
standard itself was also assessed, e.g. if this test is likely to correctly classify the target condition. In
the domain of flow and timing we evaluated the possible influence of time between index test and
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reference standard, e.g. in what order were they performed and how much time was between them.
Besides that, we evaluated the influence of patient flow, e.g. were all participants included in the
analyses, because patients lost to follow-up can differ systematically from those who remain.
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To investigate the presence of publication bias, funnel plots of the obtained effect sizes will be
visually inspected.

Data analysis
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Quantitative data were extracted to calculate effect sizes, expressed in Hedges’ g because of the
small sample sizes. All statistical analyses were performed using R 3.5.0 with R studio software
version 1.1.45323 and the ‘metafor’ package24. If the required data for effect size determination (i.e.
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mean and standard deviation of ASD and control group) were not reported in the article, data were
either requested from the corresponding author or -when possible- calculated based on other
available data (such as an F-test). Based on Cohens norms25 , we consider an effect size of 0.2-0.3 as a
small effect, 0.5 as a medium effect and 0.8 and up as a large effect.
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We performed three separate multilevel meta-analyses using a random effects model to investigate
the separate pupil characteristics (i.e. baseline pupil size, latency to constriction or dilation,
amplitude). Because several studies provided more than one effect size, a three-level random effects
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model was used26. This model accounts for three levels of variation: (1) random sampling variation,
(2) variance between outcomes within a study, and (3) between-study variance27. More details on
this method can be found in Van Den Noortgate et al. (2013)26. The model fit will be evaluated, by
checking if the full model (three levels) captures the variability in our data better than a reduced
model (two levels)28,29. A priori, multiple moderating variables, such as age, IQ, environmental
lighting, etc., were considered to investigate. However, due to the limited amount of available data,
these analyses would not have been reliable, and were therefore omitted.30 Participants’ average age
was always provided, but never at individual level. In many of the included studies age ranges were
too wide to include in a moderator variable analysis.
The qualitative analysis was conducted using Nvivo-12 software version 12.3.031. The discussion
section of included articles was coded and themes were extracted to construct an inductive analysis,
based on the Framework Method32,33. In this method, the author first familiarizes herself with the
data by reading the selected sections, the discussion section of all included papers. After that, she
starts coding the data in Nvivo, in that way extracting the themes from these sections. By doing this,
she develops a working analytical framework of how these themes can be related to each other.
Next, this framework is applied to all the data, data is recoded or charted and interpretations can be
made. The first author was the only one to analyse and code the data, therefore we did not
determine intercoder reliability (ICR) or validate our model otherwise.

The framework of these themes will be discussed in the results sections, interpretations on the
framework will be made in the discussion section.

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Results
Literature search
In total 44 studies were included in the qualitative synthesis and 41 in the quantitative synthesis (see
Table 1) according to the flow chart in figure 3. Twelve out of 44 studies investigate the pupillary light
reflex (PLR) in response to a simple light flash (or only investigate tonic baseline pupil size), the
remaining thirty-two articles reported on pupillary response to complex visual stimuli (PRC). From
now on, we will refer to these as PLR and PRC studies, respectively. Three articles34 compared infants
at risk of ASD with a control group. These articles were included in the qualitative analyses, but not in
the quantitative analyses since it only included a high-risk group of individuals who had not yet
received a diagnostic work-up. One article35 was a conference abstract with detailed quantitative
results on the outcome parameters, which we included in the quantitative analyses to avoid
influence of publication bias. However, we did not include this in the qualitative analyses, due to the
lack of qualitative content. During data-extraction, we found that six articles did not contain usable

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quantitative data on our three selected parameters. Therefore, the final amount of studies in the
quantitative synthesis is 35.

Risk of bias

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None of the studies show a ‘low’ risk of bias on all four levels, yet often the risk was ‘unclear’ instead
of ‘high’. Applicability concerns are most often low. However, because of the limited number of
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available studies, we decided to keep all studies included in the qualitative and quantitative analysis,
while being aware of their limitations in quality. The risk of bias evaluation for each individual study
is shown in table 2.
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To investigate possible publication bias, we made a funnel plot for each parameter (figure 4) and
visually inspected these for asymmetry.

Quantitative results
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Table 3 displays effect sizes for each study for baseline pupil size, latency to constriction/dilation and
amplitude of the change, with their corresponding variance and weight. When a study included
multiple effect sizes, they are all represented separately. The clustering of this data is accounted for
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by using a multilevel model. Three separate multilevel meta-analyses were performed using a
random effects model to investigate the separate pupil characteristics. Results are shown in forest
plots in Figure 5.

Effect sizes of the baseline and amplitude parameter are small and differ widely between studies,
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which results in insignificant pooled effect sizes and therefore do not show any significant group
differences for these parameters. This is shown by the blue bar of pooled effect sizes in the forest
plots in Figure 5, which cross the zero-line. However, the pooled effect size of the latency parameter
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is a Hedges’ g of 1.03 (95% CI: 0.49-1.56, p=0.0008), which is considered a large effect25. This shows
that the latency parameter has the most potential of showing group differences between ASD and TD
groups.

For each separate meta-analysis, we investigated the heterogeneity at the second and third level, to
check if the multilevel model was the best fit for this analysis. We compare our full model (three
levels) to a reduced model (two levels), where we fix a certain level to zero to remove it and fit the
model again. The Akaike information criterion (AIC) and the Bayesian information criterion (BIC) are
indices of model fit. Two separate log-likelihood-ratio tests were performed for each level. For more
detailed methods, refer to Assink and Wibbelink28. Results are shown in Table 4. For the baseline and
the latency parameters, there is significant within-study variance (level 2), indicating variance
between effect sizes extracted from the same study, but not between-study variance (level 3).
However, the amplitude parameter shows significant within and between-study variance, indicating
there is more variance than to be expected based on the sampling variance alone (level 1).
To gain insight in the proportion of these different types of variance in each meta-analysis, we
calculated the distribution of the total variance over the three different levels of variance in our
multi-level model, which is shown in Figure 629.

Qualitative analysis
The proposed explanations for the group differences in pupillary response as presented in the
discussion section of the included articles were analysed. A model for summarizing and interpreting
this data was developed via the framework analysis approach. The resulting framework model,
created based on the extracted themes found by the first author, is pictured in Figure 7. As illustrated
in Figure 7, these explanations can either be situated at the level of stimulus input, the information
processing physiological pathway, or at the output response level. Moderating factors can either be

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considered as external or subject-dependent influences. An integrative interpretation will be
provided in the Discussion section.

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Input

Several studies focused on the pupillary reflex to a simple light flash, but other studies investigated
the pupillary response during the processing of more complex visual stimuli, which can contain socio-
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emotional information36,37, compare familiar and unfamiliar information23,27, require local or global
processing39,40, and may involve active processing strategies. Differences in input cause different
information processing and therefore differences in the pupil response. Input is influenced by
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environmental factors such as lighting conditions41, which differ between studies. Fan et al. also
included several light intensities in the same participants, where the group differences in amplitude
were only significant in one light condition (DA794), and not in the others.42 They have only found a
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significantly decreased relative constriction in the ASD group in this condition, due to the generally
smaller constriction amplitudes in the other conditions, which consequently suppress the group
differences. Aguillon-Hernandez et al43 hypothesise that complex visual stimuli with a brighter
presentation than the black interstimulus slides could elicit a pupillary light reflex that interferes with
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the event related pupil dilation. This way of presenting stimuli with a predicable variation of
luminosity could even elicit a conditioned response.

Physiological processing
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- Anatomy

The physiological processing of the presented stimuli occurs in several anatomical structures.
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The pupillary response is used as a test of cranial nerve II and III41, but this pupillary pathway can be
affected in every segment. Lesions or demyelination42 can cause problems at the level of signal
transduction, which could result in a longer latency of the pupil response. Longer latency in ASD was
also reported in other neurological systems than PLR, for example in saccadic eye movements and
auditory evoked response44. The maturation of the pupillary pathway can be monitored using the
PLR41. Through neuroanatomical and neurochemical studies, larger tonic pupil size has been
suggested to reflect pathology within the pupillary system40,42. Not all interactions and modulations
by cortical and other brain structures with the PLR pathway are fully understood44. The tectopulvinar
system is responsible for detecting and orienting towards visual stimuli and the geniculostriate
system regulates visual pattern analysis. The first was not implied to have any deficits in ASD, but
evidence on the geniculostriate system remains unclear45.
Another structure that is important in the processing of the input is the cerebellum, which modulates
sensory input42. Post-mortem studies46 showed a decreased Purkinje cell density in ASD, but
morphological studies of the cerebellum have inconsistent results. Animal studies imply that the
cerebellum is involved in the pupillary light response and lesions in the cerebellum also influence the
PLR42.
The amygdala is a third structure that has been studied extensively in ASD. Nuske et al. found that
the pupillary responses of children in the ASD group to fearful expressions by familiar people were
similar to those of the TD group. However, the children in the ASD group showed a reduced
amplitude change in comparison to those in the TD group in response to fear expressed unfamiliar
people. Nuske et al. link their results to reduced engagement of the amygdala in response to fearful
expressions of unfamiliar people. The amygdala could influence the pupillary response when stimuli
elicit an emotional reaction38.
Finally, some authors also suggest top-down influences from structures outside of the reflex-arc47.

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- ANS

The autonomic nervous system is an important mediator of information processing in the brain. This
system can be influenced by age, other neural circuits, level of arousal or a cholinergic disruption and

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is relevant in the pupillary response. The cholinergic disruption was previously described in animal
data. For example, nicotinic acetylcholine receptor subunit alpha 7, which mediates the fast
excitatory synaptic transmission in the pupillary light reflex in animals, is upregulated in individuals
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with ASD48. Another hypothesis why the ANS could be an important mediator is found in the locus
coeruleus/norepinephrine (LC-NE) system41. In response to surprise or change in the environment,
NE is assumed to change cortical gain and furthermore, NE antagonism selectively impairs volatility
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learning, which leads Lawson et al. to conclude, based on their results in adults with ASD, that
atypicalities in the LC-NE system may be present in ASD.49 Cortical gain increases behavioural
flexibility, exploration of the task environment and as such to relevant and irrelevant stimuli. When
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phasic responses decrease, the focus shifts only to task-relevant stimuli, according to Granovetter et
al.50. In their study, the ASD group showed inflexibility of regulation of the LC activity, they conclude
based on a lower pupil reactivity in the presence of distractions, which is not expected when the LC
would be regulated correctly. In toddlers with ASD, this LC-NE system is more often in a phasic state,
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which could be advantageous in a visual search task as performed by Blaser et al. and could be a
starting point for other cascading ASD characteristics51
The LC provides NE to the amygdala, thalamus and superior colliculi which are implied to be part of
the low frequency face detector system, which would provide an automatic face processing.43 This
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system could be impaired in ASD hypothesise Aguillon-Hernandez et al.43, because of their findings of
reduced reactivity to faces. Another structure receiving NE from the LC is the hippocampus, which is
important in memory52. Alterations in this neurotransmission may lead to reduced consolidation and
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retrieval in ASD and thus result in reduced differences in pupillary responses to new versus old items
compared to a TD group.52
Phasic LC-NE activity and related pupil dilation has been linked to sensory-perceptual processing,
orienting attention and to social cognition.53 As described in the introduction individuals with ASD
might have an altered neuroception of safety versus threat which results in elevated autonomic
arousal and is also suggested by Del Valle Rubido et al. as an explanation for the between-group
differences in their study.54
Changes in the various pupillary parameters in the PLR are often not correlated to each other, which
suggests influences of different neurological mechanisms according to Kercher et al55. Pupil size, both
baseline size and size after responding to a stimulus, may be under sympathetic control, but speed of
constriction is dependent on the speed of the contraction of the iris muscle, which is under
parasympathetic control. Latency represents signal transduction, thus might reflect synaptic
function, white matter maturation or network connectivity55.
Wagner et al.56 showed that in infants at increased likelihood for developing ASD (HRA), pupil sizes
are larger in response to emotionally-salient faces and based on previous studies confirming that
larger pupils in response to this type of stimuli are related to an activation of the SNS, they conclude
that in these HRA infants the SNS is more activated than in the TD infants. Anderson et al. state that
previous research showed that β-antagonists such as propranolol led to improvements in aggression
and anxiety, amongst other symptoms, by influencing cortical NE-modulated connections.57
Heightened autonomic responses could reflect increased arousal and stress, which could be caused
by the testing experience and environment40.

- ANS - bodily functions

The autonomic nervous system effects upon many other bodily functions besides the pupillary
response, such as the heart rate, which on average is increased in the ASD group58. According to the

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read studies, increased heart rate in the ASD group also correlated with sensory processing
symptoms in this group58, as measured by some items of the Sensory Profile59, such as avoiding
getting messy or going barefoot, or expressing distress during grooming. Heart rate variability

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changes are smaller in the ASD group during the PLR-testing, albeit possibly influenced by the slightly
forward inclining position during the PLR-testing60. In addition to heart rate, the ANS also affects skin
conductance. Sweating increased when pupil size was larger, due to a higher sympathetic tone, in the
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TD group, but not in the ASD group, which indicates a possible disruption of normal sympathetic
control in the children with ASD44. Other bodily functions that are influenced by the ANS, such as
blood pressure, respiration rate, and heart rate have been found to be correlated with body mass
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index (BMI) in the TD group, but to date this has not been shown for pupil response40, or for the ASD
group. Anderson et al. investigated the link between salivary alpha-amylase (sAA) and NE, even
though this relation is not yet proven definitively. They found larger tonic pupil size and lower
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afternoon levels of sAA in the ASD group, which could both not be explained by stress, because they
controlled for this by measuring salivary cortisol. sAA could be an alternative biomarker in the
research on the ANS.57

Subject-dependent influences
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- Effect of age and development

The presence of an ASD vs TD group difference in PLR response seems to be age-dependent. An age
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trend was observed by Dinalankara44, and Nyström47 extended this trend. In children from two to six
years of age, the group difference in the latency parameter increases when they grow older and is
smaller when they are younger. Nyström observed that in the younger age group this difference
seems to have flipped: latency is longer in the TD group than in the ASD group. Kercher et al.55
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confirm this based on one child with an early diagnosis of ASD in their sample, which has the shortest
latency of their entire HRA infants group at 12mo and increased at 18mo. Daluwatte et al.60 also
confirmed this effect of age on the latency parameter in the TD group, but could not find this effect
in the ASD group.
Dinalankara et al.44 also noticed an age trend when investigating baseline pupil size. In TD children,
the baseline pupil size increased with age, as previously studied, but this increase was not observed
in children with ASD. The two trajectories intersected at the age of four years old: beneath this age,
children with ASD had a larger mean baseline pupil size than TD children, and above this age, TD
children had a larger mean baseline pupil size. A possible explanation for these age trends could be
found in white matter maturation, which is accelerated in ASD44. Kercher et al.55 confirmed an
increase in baseline pupil size with age in TD children, as well as a decrease in latency and an increase
in relative constriction. Nyström et al.34,47 found a larger constriction in the HRA infants at the age of
9-10mo compared to TD infants, but Kercher et al.55 found the opposite at group level, which they
attribute to differences in methodology and testing conditions.
Rubin found an age effect in his 1961 study61 between TD children and TD adults and speculates that
hormonal changes in puberty could possibly have an influence on autonomic responsiveness.
Immaturity of perceptual processing can influence task results with complex stimuli in younger
subjects.62 In children with ASD, specific interests in objects are more present at a younger age, so
younger children might have different moral judgments and stronger pupillary responses to damage
to objects than older children.63
In many of the included studies, the participants’ age range is wide, so there are not enough
participants in the same small age interval to differentiate between intervals.

Heritability of ASD is high. An association between genes and pupillary responses has not yet been

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made, but Nyström suggests that shared environmental influences in ASD are limited and the results
of his study thus reflect the effect of genetically mediated differences48.

- ASD traits – socio-communicative domain

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Results are also influenced by traits that are inherent to the ASD diagnosis. The socio-communicative
domain is studied intensively in observational studies of social behaviour, but pupil responses can
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also be of interest when investigating this domain. This was shown by Martineau64, who investigated
the PLR waveform in response to stimuli of varying social importance, i.e. human faces, avatars, and
objects. While amplitude change in response towards faces clearly differed from responses towards
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avatars and objects within the mental age matched TD group, responses were undifferentiated
within the ASD group. However, the responses of the ASD group did not differ significantly from the
mental age matched TD group. Aguillon-Hernandez et al.43 found that only dilation in response to
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virtual faces was stronger in ASD than in the TD group, and further confirmed the undifferentiated
responses in the ASD group that were also found by Martineau.65 They hypothesise that dynamic
emotional faces might elicit a larger response than static ones because of the larger emotional
contagion in a dynamic or action context.
Individuals with ASD often have more anxiety for unfamiliar people or situations than TD individuals.
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In Nuske et al.66, the ASD group showed greater pupil dilation than the TD group in response to
viewing unfamiliar people. Previous evidence points to larger SCRs in ASD and TD children to familiar
compared to unfamiliar people, so we would expect the pupil to increase when the SCRs increase in
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response to the familiar people rather than the unfamiliar people. More pupil dilation to direct gaze
and averted gaze in familiar people and more pupil dilation to mutual gaze in unfamiliar people was
related to less internalising symptoms in the ASD in the same study by Nuske et al.
Reactivity to others’ emotions differed in the ASD group compared to the TD group in another study
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by Nuske in 201438: a larger amplitude in response to fearful faces of familiar people were associated
with fewer communication and play deficits. In the task with familiar and unfamiliar stimuli Nuske et
al. found that visual attention was correlated with peak pupil dilation amplitude in response to
familiar faces, in the ASD group only when they paid attention to the eyes, and in the TD group only
when they paid attention to the mouth 38. Wagner et al.56 showed that in infants at increased
likelihood of developing ASD there is a negative association between 9-month pupil size in response
to faces and 18 month social-communicative outcome.
Aldaqre67 investigated the pupillary response in relation to the differential processing of pointing
versus grasping gestures. There was no significant difference in pupil dilation between ASD group
and TD group overall, which shows intact social sensitivity. Within the ASD group, the difference in
pupil dilation amplitude to the pointing and the grasping cues was larger than within the TD group,
which could be explained by the higher communicative value of the pointing stimulus. Aldaqre
suggests that understanding the gesture could be promoted by performing the same gesture, which
is known to be delayed in ASD, and could cause the increased mental effort, which could explain the
within-group difference. In 2006, Anderson39 reported pupillary constriction in the ASD group when
looking at the internal feature region of the face (eyes, nose, mouth), compared to pupil dilation in
the TD and delayed group. He suggests the pupil response could be a more sensitive predictor of ASD
than scanning responses. Falck-Ytter68 showed that individuals with ASD looked more to same
features of the face during upright and inverted presentation. The ASD group showed more pupil
dilation during inversion, where the TD group did not, which could be a sign of enhanced processing
of face features in ASD, requiring more mental effort.Dicriscio69 investigates her participants in a
dimensional way, and shows inverse relationships between autism traits measured via the Social
Responsiveness Scale (SRS)70 and amplitude of pupil dilation.
Müller et al. 71 found a decreased pupil dilation in ASD as compared to TD, while watching a movie

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for the assessment of social cognition. Since the pupil dilation did not differ during the introductory
slides, they believe this effect is caused by the social aspect of the stimulus. Reduced pupil dilation in
ASD for processing social content can, according to Müller et al, be explained by the low social

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motivation theory, which states that people with ASD have a diminished reward value for social cues
in the frontostriatal network.
Hepach et al.72 found a discrepancy between the cognitive and affective aspect of prosociality in the
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ASD group. Pupil dilation, considered the affective aspect, was similar in ASD and TD groups, but the
anticipatory gaze of children in the ASD group did not lean towards to correct prosocial answer,
whereas the children in TD group did show this preference.
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In ASD, play can be impaired at an early age. Nuske et al. found that in the ASD group, deficits in the
area of functional and symbolic play were negatively correlated with pupillary responses in the
longer conscious emotion exposure (2 s) condition. Children who had a more typical magnitude of
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physiological response to conscious emotion exposure had fewer deficits in the area of play36.

- Processing of emotional information

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Emotional processing can be aberrant in individuals with ASD, compared to TD37. Reactions to
emotional faces have been shown to be altered in ASD. For example, Nuske et al. showed that
unconscious emotional reactivity was altered in the ASD group, with larger responses to neutral faces
than the TD group, and a larger response to neutral than to fearful faces36. Wagner et al.56 found the
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opposite in infants at risk of developing ASD: they show a larger pupil dilation to emotional faces
than the low risk group. Children with ASD showed no increased reactivity to dynamic emotional
faces in the study by Aguillon-Hernandez et al.43, in comparison to static stimuli within ASD, and this
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discriminated them from the TD group. On the contrary, Galazka et al.73 found pupillary contagion in
ASD, similar to TD, and similar in response to different emotions.
Sepeta et al.74 hypothesise that their results, increased pupillary responses in response to happy
faces in the TD group and not in the ASD group, can be explained by an impairment in social reward
processing in ASD, since attractive faces with direct eye-contact are known to provoke activation of
the ventral striatum, which is part of the neural reward processing system. Reisinger et al.75 found
the opposite effect, increased pupil reactivity in the ASD group compared to TD. In another article by
Nuske et al.38, the children with ASD had reduced pupillary responses to the fearful expressions of
unfamiliar people, relative to the typically developing children. The ‘gaze aversion hypothesis’, which
states that individuals with ASD have a hyperaroused response to direct gaze, has also been tested
with pupillometry by Nuske et al, and they found no evidence of aversion to direct gaze in the ASD or
TD group. ASD and TD both had a greater pupil dilation to direct versus averted gaze. Sepeta et al.74
also argue against this hypothesis, since their study shows similar pupillary responses in TD and ASD
when watching a fearful or anger face with a direct gaze, as do Reisinger et al.75. Galazka et al.73
found that the ASD group looked less at the eyes in their study, but despite this, showed similar
pupillary contagion as the TD group. In 2016, Nuske further investigated emotional learning in ASD37.
TD children, but not children with ASD, learned about the emotional value of objects via vicarious
emotional reactions. In the ASD group, there were no significant differences in pupil dilation before
and after viewing the emotional stimulus, where in the TD group there was an increase. In the ASD-
group, the social-emotional calibration to a happy stimulus was associated with overall ASD severity,
suggesting increased ASD severity is related to increased difficulties in aligning to positive emotions
of others. Difficulties in social-emotional calibration could potentially explain idiosyncratic emotional
reactions to sensory features of the environment, which are often present in ASD37.
Krach et al.76 investigated the fMRI and pupillary response to watching social and physical pain and

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found that in ASD, the social pain is processed differently than in TD (more hippocampal activation
during self-report), where as in the physical pain condition there are no group differences. The
complexity of the situation and motivational aspects might impact the response.

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- Moral reasoning

Garon et al.77 found that during moral dilemmas pupil dilation in ASD and TD groups is similar, even
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though individuals in the ASD group seemed to have more difficulty understanding the dilemma and
produced fewer socially adaptive moral decisions. Moral judgements were made similarly by children
with ASD and the TD group in the study by Li et al.63 and both groups showed similar increases in
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pupil dilation in response to moral reasoning, however some patterns in the ASD group were
remarkable. The children with ASD evaluated damage to an object as worse than damage to a person
and this was also reflected in larger pupil dilation to damaging an object, which might be an effect of
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a special interest in object, according to Li et al.63

- Relationship with RRBI’s

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A characteristic of ASD within the domain of repetitive and restrictive behaviours (RRBI’s) is cognitive
perseveration. Gotham et al.78 state that this perseveration is similar to depressive rumination.
Individuals with ASD who had elevated rumination had similar pupil response patterns to sad stimuli
as depressed TD adults, as well as elevated depressive symptoms78.
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Another ASD related trait is insistence on sameness. This possibly influences the reaction to
incongruency. Aldaqre et al.67 found an overall increase of pupil dilation for the incongruent
condition in both groups; the saccadic reaction times did differ so different cognitive sources might
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be involved.
In Sepeta et al.74 the happy faces might have been an unexpected stimulus for the ASD group, if they
had built up a prior expectation of negative facial emotions, which could consequently cause a pupil
dilation through recognizing this as a new target in the environment, in which the LC also plays a
part.
Lawson et al.49 demonstrate that adults with ASD show less distinctive responses to expected or
unexpected stimuli than TD adults, whose pupils dilate significantly more to unexpected stimuli than
to expected stimuli. Reduced reliance on perceptual priors was also studied Laeng et al.62 by
investigating the pupillary response to illusory brightness, where pupils would constrict because they
expect an increase in luminance. Laeng et al.62 hypothesized this constriction would be reduced or
absent in the ASD group, but found no differences between the ASD and the TD group.
The inability to distinguish relevant from irrelevant stimuli as found by Granovetter et al.50 impedes
the creation of priors and expectations and the ability to learn from novel environmental input and
consequently could cause increased attention to particular environmental stimuli that other don’t
consider relevant and this explain the fixated interests in ASD, as well as increased sensitivity to
sensory stimuli. They confirmed this in their results, as a smaller pupil response amplitude, which
shows low phasic LC activity and increased cortical gain, was associated higher ADOS-RRB scores.50
Specific interests are common in ASD. Traynor et al.79 found increased pupil dilation in the ASD group
to circumscribed stimuli compared to social and neutral stimuli, showing increased motivation which
results in increased autonomic arousal.

- Altered Sensory Processing

Sensory processing is often altered in ASD, as hyper- or hyporeactivity to sensory stimuli is a core
characteristic of the second domain of ASD. Daluwatte58 found that in the ASD group, children with
more atypical sensory behaviours also had smaller PLR constriction amplitudes. When investigating

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these sensory scores in more detail, it was noticed that children with ASD who reported ‘‘never’’ on
five sensory items (which means less sensory issues) had greater PLR constriction amplitude than
those who reported ‘‘always’’. Nyström et al.48 also categorize the hypersensitive pupillary light

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reflex in infants at risk for ASD within the known sensory abnormalities in ASD. Furthermore, they
extends the findings by Daluwatte et al.58 by suggesting that atypicalities in neural systems involved
in sensory processing could play an important role in the early development of ASD. Daluwatte et al.
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question why only the change in amplitude seems to be implied in sensory abnormalities, and not
latency of this change. They hypothesise that both pupillary amplitude and the sensory behaviour
assessment measure quantitative differences at one point in time, in comparison to latency, which
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measures a speed or duration over multiple points in time.
Richey et al. 80 hypothesise that an impairment in sensory integration, which is often present in ASD,
could potentially influence their results, as they combined visual stimuli with auditory instructions.
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- Altered perceptual processing

Individuals with ASD sometimes have an advantage over the typically developing population.
Toddlers with ASD have a greater attentional focus, according to Blaser51, because of differences in
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the LC-NE system. In his study, the ASD group has greater phasic pupil response during visual search,
and larger phasic pupil response is associated with better search performance. Different ways of
focussing attention, e.g. adjusting focus of visual attention to either brighter or darker areas, could
have a differential effect on pupil size, correlated with scores on the Autism Spectrum Quotient in a
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TD population.62
Another possible advantage of individuals with ASD is in the area of perceptual differences.
Anderson’s study in 200639 showed a significant decrease in the ASD group in visual scanning to
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landscapes, which consisted of ‘local’ elements without a dominant ‘global’ configuration. In

addition, the ASD group showed pupillary constriction to children’s faces, while control groups
showed pupillary dilation. Visual scanning responses to landscapes had a negative correlation with
the Behaviour subscale of the ADOS-G for the ASD group, meaning that in this group briefer fixation
time is correlated with less behavioural symptoms of ASD. The pupillary responses in this study did
not correlate with the ADOS-G scores.

- Cognitive functioning, mental effort and attention

Cognitive factors also influence the pupil response. In psychological research the pupil has been
extensively researched as an index of cognitive effort39. A cognitive reappraisal task by Richey et al.80
showed no group differences in pupil dilation, thus concluding that it took similar effort in the high
functioning ASD and the TD group. Müller et al.71 consider the diminished pupil dilation in their
results as a decreased processing effort.
Attentional focus could increase pupil size51, but this seems most relevant for demanding tasks, not
passive viewing tasks62. Delayed orienting of attention could be reflected by a delayed latency in the
study by Bast et al., which would be in agreement with the known delayed orienting in ASD in
behavioural tasks53. Boxhoorn et al.81 suggest increased pupil dilation is a result of a persistent
hyperphasic state, with enhanced attentional focus, but also reflects increased task difficulty or
cognitive load, which as a result leads them to interpret increased pupil dilation in the ASD group in
their study as a higher effort to control the focus of attention during reflexive orienting in ASD. Del
Valle Rubido et al.54 suggest pupil size might reflect the ability to better arrange effortful attention
and interpret this as a sign of greater cognitive or inhibitory control, even though this appears to be
inconsistent with their own results.

Most research participants have an FSIQ of 80 or higher or an (at least) average performance on the

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Mullen Scale of Early Learning (MSEL) and are therefore considered participants with an average or
higher intelligence. Dicriscio et al.69 included a wide range of IQs in her study with a dimensional
approach. Full scale IQ was correlated with pupil dilation amplitude (r=0.31) and SRS Total T-score

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(r=0.55). Pupil dilation amplitude and FSIQ could significantly predict the SRS total T-score, in a model
based upon these results. In the study of Del Valle Rubido et al.54 better functioning, defined as less
hyperactivity and higher FSIQ and PIQ scores, was related to larger pupil sizes in the ASD group.
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Reisinger et al.75 included a cognitively diverse group. In their sample, cognitive functioning was not
impairing overall attention during the task. However, social attention profiles specific to cognitive
abilities need to be further explored.
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In the results of Hepach et al.72 the developmental quotient (DQ) modulates the level of arousal, with
low-DQ children having the greatest arousal in the TD group, but high-DQ children having the
greatest arousal within the ASD group.
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External factors

Medication is an external factor that influences the information processing and neurotransmission.
Individuals with ASD in the different studies take various types of drugs that can influence this
processing at various levels. In some articles, medication-use was an exclusion criterion.
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Daluwatte et al. found a correlation between PLR constriction and sensory score in the ASD group
who did not take medication, but not in the remaining ASD group who did take medication or in the
TD group58. Daluwatte suggests that it is likely that medication does affect the ANS function in a
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subtle way, even though medication did not show a significant effect on either sensory scores or PLR
parameters58. Granovetter et al. included medication use in their model, they even investigated
adrenergic-related medication separately, and found no effect on their results.50 In other studies,
medication effect is either not present, or only in a group with low functioning ASD, where it
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complicates the relation between IQ and PLR latency outcomes. Daluwatte et al, for example, state
that lower functioning participants may require more medications because of more severe
symptoms60. Anderson et al. argue that previous research showed that β-antagonists such as
propranolol led to improvements in aggression, irritability, anxiety, self-injurious behaviours,
increased sleep time, task performance and working memory, because this medication reduces NE-
modulated connections within cortical regions responsible for higher-order processing of
communication and social responsiveness.57
Reisinger et al. suggest their paradigms could potentially be used in the future to monitor treatment
effect of treatments targeting social and emotional impairments, following a clinical trial in Fragile X
using an extended version of the current trial.

Discussion
This systematic review and meta-analysis provide insights into the value of pupillometry for
measuring differences in ANS functioning in individuals with ASD and typically developing peers. Our
quantitative results confirm a robust difference between these groups in the latency of the pupillary
response. There are no significant group differences for amplitude and baseline of the pupillary
response. When clustering qualitative evidence on the underlying hypotheses of these group
differences, we came across many explanations given by the authors of the included papers. Through
framework synthesis, we constructed the model in Figure 7. When critically reflecting on this
evidence, we come across many potential pitfalls. In this review, we grouped these experiments
despite their differences, to evaluate the entire field. This results in a heterogeneous set of
information. Across the various studies, hypotheses were not always clearly defined or sufficiently

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substantiated, which resulted in a multitude of explanations included in our qualitative framework.

On the level of input, we notice that the study designs included in this review varied widely, both in
the set-up, e.g. equipment used and environment adapted, as well as the experiment itself, by using

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a light flash or a more complex stimulus. For example Fan et al. 42 conducted the same experiment in
different lighting conditions and found different results in terms of group comparisons, which shows
the large influence this parameter can have. If the pupil reacts already varyingly to a stimulus as
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simple as a light flash, we should be careful when interpreting the pupillary response to more
complex stimuli.
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Various anatomical substrates are implied to have an influence on the pupillary response. The ANS is
connected to many other structures, such as the limbic system. These connections can vary during
different stages of development and therefore could be of importance when interpreting our results.
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The current pupillometry studies do not allow to distinguish separate influences from different
origins, since most of the studies only included measures of the pupil and gaze of the participant and
these findings were not corroborated with other measurements such as cardiac monitoring,
respiratory monitoring, sAA or skin conductance. These additional measurements would also inform
us about the role of the sympathetic and parasympathetic responses of the body and how this is
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reflected in the peripheral NS, as for example was done by Bradley et al in 2008, showing a
covariance between responses of the pupil and the skin conductance to emotional stimuli in typically
developing adults82. In most studies, the parasympathetic and sympathetic systems are not
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investigated separately. Knowing how intertwined these two systems are in the pupillary response,
makes it hard to state anything about these systems separately. Combining different measures could
help us gain more insight into balance of the ANS in the central nervous system. This balance is
complex and influenced by many other pathways and structures. Directionality of effects within the
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LC-NE system are not always obvious. A parasympathetic deficit in the ASD group could be the basis
of the differences measured with pupillometry. Nevertheless, we cannot exclude a sympathetic
increase as a possible causal factor. Reimer et al. have shown that both pathways are coupled to
pupil variations in mice, but the timeframe of the response differs slightly. Before the peak of
dilation, norepinephrine activity levels were larger and had a shorter latency than acetylcholine
activity. In addition phasic activity in noradrenergic axons was associated with rapid fluctuations,
while tonic cholinergic activity caused longer-lasting dilations of the pupil.83 While growing evidence
suggests that the most likely explanation for group differences lies in the LC-NE system, we cannot
conclude this solely on pupillometry data, considering the many different influences on this pathway
we discussed earlier.
While most authors point to the autonomic nervous system as an underlying cause for these group
differences, we wonder why this would affect latency rather than baseline pupil size or amplitude
change given that all these parameters are under the influence of both the parasympathetic and
sympathetic nervous system. Consequently, we cannot confirm nor contest the polyvagal theory in
ASD based on the current evidence in pupillometry.
It would also be useful to compare our included populations with individuals with ADHD. Bellato et
al84 show hypo-arousal in the ADHD group as measured by SCR and HRV, but in contrast the only
pupillometry study that was included in this review, did not show any differences in ANS functioning
between ADHD and TD. This contrasting finding therefore again emphasizes the importance of future
research with combined measures of the ANS, as discussed earlier.

A developmental aspect was also suggested by multiple authors. Yet, in most articles the included
age range was wide, so within a study there were not sufficient participants in the same age interval
to robustly demonstrate age effects. Dinalankara44 did observe an age trend, as described in the

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result section. This developmental aspect should be further investigated, preferably in a longitudinal
study of infants and young children. After data-collection for this meta-analysis, a paper by Kercher
et al. was published, which confirmed a developmental effect and group differences between infants

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at increased likelihood of developing ASD and TD infants from the age of 6 months to 24 months.
These different developmental trajectories were not only found when investigating the latency
parameter, but also on the level of baseline pupil size and amplitude change55. Reproduction and
extension of these findings is instrumental. -p
Maturation of the white matter was given as one of the possible explanations of this observed age
effect. Ben Bashat et al. demonstrated accelerated maturation of the brain of toddlers with ASD in
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2007. The exact cause of the increase they found is still up for debate: it could be caused by increase
in number, size of axons or myelination processes, or it could be a result of decreased pruning.85
Hoppenbrouwers et al. also report a developmental switch in relation to white matter maturation in
the ASD group in their meta-analysis, with findings of hyperconnectivity at an early age and
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hypoconnectivity in older children86. The relationship between white matter and the pupillary
response could be found in the importance of white matter in signal transduction. The pupillary
response, and especially the latency of this response, represents the speed of neural signal
transduction87.
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We suggest that maturation of other brain structures could also influence ANS functioning. For
example, the limbic system is closely intertwined with the ANS in early development88, which could
have an influence on the pupillary response. It would be useful to investigate the pupillary response
of individuals born prematurely, because the parasympathetic system develops in the final weeks of
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the pregnancy and could be influenced by early delivery89. Extreme preterms also have an elevated
chance of developing ASD, which makes this an even more interesting group to investigate90.
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Social-communicative sensitivity was measured by investigating the pupillary response. We wonder

whether our investigated three pupillary response parameters are the best indices to measure this
sensitivity since many other options exist: from behavioural observations to questionnaires or EEG
measurements. Though the pupil is a quite simple index to measure, many authors use this as an
index of various complex constructs, ranging from social-communicative sensitivity to emotional
processing, decision-making or effort91,92.

Gotham et al.78 assume that in the RRBI’s domain, perseveration and rumination are related. This can
be questioned, however. Perseveration in ASD does not have to contain an affective component,
while rumination does93: it can be associated with depressive thoughts and or feelings of anger. This
affective component is related to arousal94 and therefore it is important to take it into consideration
when studying the pupillary response. According to Patel et al.93 rumination in ASD is not only due to
increased perseveration but also to decreased inhibitory control. It has been shown that individuals
with ASD have poorer inhibitory control95, so this factor should be taken into account. Gotham uses
various measures to quantify symptoms in the RRBI domain of ASD, but doesn’t specifically measure
perseveration separately and does not consider inhibitory control. Patel et al. show that anger
rumination is associated with dysregulated emotions and behaviour in ASD93, which is also not
measured by Gotham, but could be relevant to include in future studies. When summarizing different
opinions on these concepts, we question what the pupil can actually represent. The pupillary
response can be specifically due to the affective component of rumination, which causes arousal,
rather than to the perseveration that is typical in ASD.

Very few studies report adequately on sensory functioning, though in typical development, this is
influenced by the level of arousal of the ANS96. It would be interesting to take sensory hyper- or
hyposensitivity into account, for example measured by the Sensory Profile59. Rogers and Ozonoff10

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conclude that evidence on the link between sensory functioning and arousal is inconclusive and often
of limited quality, but that across all sensory domains, more evidence supports a theory of
physiological hypo- than hyper-responsiveness in children with autism.

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When exploring the cognitive abilities of the included participants, we notice that most studies focus
on high-functioning individuals with ASD. This makes the conduct of eye-tracking easier, but it would
be interesting to evaluate whether ANS functioning is also altered in lower functioning individuals.
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The pupil can represent cognitive effort91, which is not always taken into account when designing a
study, specifically with more complex stimuli. Attention is inherently linked to the ANS, so it seems
necessary to take into account in all pupillometry studies.
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The pupil can reflect a response to an emotional stimulus, but this reflects the arousal caused by this
stimulus.97 Oliva and Anikin investigated emotion recognition, by letting participants make a decision
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about emotional state based on nonverbal vocalizations. Their results suggest that decision-making
influences the time course of the pupillary response in a typically developing sample. It would be
interesting to extend this finding to ASD and investigate differences in this population.

External factors such as certain patient characteristics (e.g. medication use) and environmental light
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should be closely monitored and reported in future studies, to investigate whether these factors
could be moderating. For example, antipsychotics, which are frequently prescribed to individuals
with ASD, can dilate the pupils through their anticholinergic side effects.98 This anticholinergic effect
is a greater issue in older generation antipsychotics98,99, so we recommend to investigate the effects
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of different types of medication on the pupil in the ASD and the TD group in future research. This
knowledge could help clinicians in deciding which antipsychotic to prescribe, if they can take the
sensitivity of the ANS into account, before possible side effects occur.
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When reflecting on the quantitative results, we observe a large amount of heterogeneity in both the
baseline pupil size parameter and in the amplitude change parameter. This heterogeneity could
reflect either clinical heterogeneity or statistical heterogeneity. Individuals with ASD can present
with an extensive range of different behaviours and characteristics, despite all receiving the same
diagnosis. This can be due to different aetiologies of ASD, either genetic, environmental or a
combination of both. Underlying differences in ANS functioning can be present. Looking for subtypes
or endophenotypes within this heterogeneous group could help clinicians to learn more about
distinct developmental trajectories or possible arousal-related comorbid symptoms, such as anxiety
or sensory hypersensitivity. Statistical heterogeneity was shown through our analyses of variance,
but we could not pinpoint the underlying cause. The particular definition of the parameters differs
between the studies. Latency to constriction, for instance, can be defined as the time between
stimulus and start of pupillary constriction, between stimulus and maximum amplitude change or
between stimulus and time of absolute maximum acceleration. Baseline pupil size can be measured
at the exact moment of the stimulus, or a few milliseconds before, which is important to take into
account when comparing these measures.
Despite these differences, the latency parameter still shows a robust difference between the ASD
and TD groups. We need to be cautious when interpreting effect sizes. Cohen’s norms do not
automatically apply. As explained by Durlak in 2009100, we should interpret current findings in their
appropriate context. We should consider the quality of the source, make comparisons across similar
research conditions and consider the findings’ clinical or practical significance.100 Nevertheless, even
against this background, the observed effect size for the increased latency of the pupil response in
ASD (i.e. g = 1.03) is impressive, and may eventually open up an avenue for potential future clinical
applications. We can only speculate on why only the latency of this response differs significantly. A
prolonged latency of a response in ASD is also found in other types of responses, such as the N170

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response to faces in EEG or in auditory processing measured by MEG. 101,102 Besides altered brain
maturation, which is discussed above, we hypothesise that signal transduction at the level of
neurotransmission is altered in the ASD population11, which is reflected in latency, being a time-

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sensitive parameter, rather than in the amplitude or baseline pupil size, being more sensitive to the
entire amount of neurotransmission. Previous research shows alterations in the cholinergic
transmission103–106 that might explain the delayed latency. Pupillometry can measure the time-course
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of the ANS-mediated pupillary response very closely, in comparison to other types of ANS
measurements, such as skin conductance, which measure the peripheral nervous system response
and therefor react slower, with latency to onset of the response typically being one to two
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seconds107, which may explain why this difference in latency to peak of the response was not
detected in other studies measuring the ANS in ASD.

Limitations
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Study designs and set-ups differed between studies, as did the included populations. The amount of
included studies is limited, and their quality is disputed, which implies that we should interpret our
results with caution. We did not perform moderator variable analyses even though this would have
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been interesting, especially for age. Most studies included a wide range of ages and did not provide
results for separate age categories, making it impossible to investigate developmental changes in our
meta-analysis. Data on populations with lower intellectual or adaptive functioning is limited, so we
cannot extrapolate our findings to these populations.
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The qualitative analysis was performed solely by the first author, thus making it impossible to
investigate intercoder reliability (ICR), even though this could be an indicator of quality of qualitative
research, as suggested by O’Connor and Joffe108. An argument against ICR is that the role of the
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qualitative researcher is not to reveal objective facts, but to communicate and interpret different
perspectives present in the field.108 When not implying to represent the only true reality, as
quantitative research does, but merely an interpretation of the reality, the ICR in qualitative research
is less indispensable than in quantitative research.

An inductive analysis was used, by which we constructed a framework based on themes extracted
from the included evidence. Based on our current findings, a deductive analysis (e.g. starting from a
theoretical perspective on the LC-NE system, which most authors point towards, and looking for
evidence to test this theory) could be an interesting alternative or addition to the current analysis.33
This meta-analysis only focused on pupillometry, therefore we cannot compare this measurement
directly to other measurements of the ANS. It would be very interesting to broaden the scope to
different measures of the ANS such as heart rate variability or skin conductance response, to get a
more complete overview of the available evidence.

Future studies
The included studies had wide age ranges and included mostly high functioning individuals with ASD.
Researchers should also investigate this phenomenon in lower functioning individuals. Furthermore,
the age ranges should be narrowed in future studies, to potentially evaluate the maturation of the
ANS by investigating these parameters at a more specific age, or in a longitudinal way over time. If
sample sizes are large enough, a linear regression based on age could be attempted. Another
advantage of larger and more diverse samples would be the possibility to look into possible
endophenotypes within the ASD group.
Individual patient data meta-analysis of the current included studies would also be interesting.
However, to establish a satisfactory quality, study designs of included studies need to be optimized

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and reported thoroughly. To investigate the specificity of the atypical latency PLR response in ASD, it
will be important to also include other comparison groups such as ADHD or ex-preterm infants.

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Competing interests: The authors have declared that no competing interests exist.
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Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
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Acknowledgements
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We want to thank all the authors of the included studies who contributed their data, even if this was
not initially fully reported in their article and thus provided upon request. Furthermore, we wish to
thank Wim Van den Noortgate for educating the first author in meta-analytical methods. We also
na

thank Sarah Kips for her contribution to the initial literature search.
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Figure Captions:

Figure 1: Structures involved in the pupillary response to light. 1) Lens, 2) Retina, 3) N. opticus, 4)
optic chiasm, 5) Nucleus pretectalis, 6) Nucleus Edinger-Westphal, 7) Pre-ganglionic parasympathetic
fibres, 8) Ciliary ganglion, 9) Post-ganglionic fibres, 10) Iris – ciliary muscle.

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Figure 2: Pupillary waveform in response to light stimulus. X-axis: time, Y-axis pupil diameter.
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Figure 3. Flow chart of literature search.

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Figure 4. Funnel plots for visual inspection of publication bias. A) Baseline, B) Latency, C) Amplitude.
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Figure 5: Forest plots with effect sizes (ES) of the A) baseline, B) latency and C) amplitude
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parameters.
Red: response to light (PLR), green: response to complex stimulus (PRC), blue: pooled effect size.
Multiple effect sizes per study were included, Y-axis shows outcome number, corresponding ES,
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variance and confidence intervals can be found in table 3.

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ur
Jo
 of
ro
-p
Figure 6. Distribution of total variance over the different levels for each parameter. Level 1 =
sampling variance, level 2 = variance within studies, level 3 = variance between studies.
re
lP
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Figure 7. Framework synthesis: themes extracted from discussion section of included studies.
 Tables:

f
Reference ASD-Group Control group Stimulus Set-up Results (baseline, latency, amplitude)

1 Rubin et al, 1961, PLR
Patterns of pupillary
dilatation and
constriction in
psychotic adults and
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5 ASD children in total. 4 no-ASD children in total. Dark adaptation, then 10s Grass camera 150mm lens and * Baseline: ASD smaller baseline pupil than normal children.
Range (months): 84 – 144 Range (months): 84 – 144 intense adapting light in electronic flash. * Amplitude: Rate of constriction significantly greater in normal
3M/2F 3M/1F subject’s right eye. children than in ASD.
75W Mazda lamp to light- * Latency: /
Diagnosis: ? 23 normal adults adapt eye to constant

autistic children61
2 Van Engeland et al, PRC
1990, Abnormal
Electrodermal
Reactivity to Novel
pr
intensity.
20 ‘nonretarded autistic’ children 20 ‘normal’ children in total. Stimulus at 60 cm, 2 sec. Whittaker Eye View Monitor * Baseline: No difference
in total. Mean age (years): 10.1 Habituation and Recovery (EVM 1998 S), corneal * Amplitude: Pupil constriction reaction decreased over time,
Mean age (years): 9.7 11M/9F condition: reflection technique. no effect of group
17M/3F Half: 18 stimuli with 4 bits of LSI-2-20 minicomputer for * Latency: /

visual Stimuli in
Autistic Children45
Diagnosis: DSM-III criteria for
‘infantile autism, full syndrome
present’.
Review of prior records, child
e-
20 children with internalizing information in the middle of sampling of horizontal and
disorder the screen, followed by 18 in vertical eye position and pupil
Mean age (years): 9.5 the right upper corner of the diameter.
16M/4F screen. Other half: same with EVM computer program.
60bits. Dentist’s chair in an

psychiatric observation,
assessment of nonverbal
intelligence WISC-R, assessment of
autistic symptomatology CARS.
Pr
20 children with externalizing Counting condition: 36 stimuli, acoustically and electrically
disorder consisting of 15 X’s and 21 O’s shielded room.
Mean age (years): 9.6 delivered at various locations
15M/5F on the screen for 2 seconds.

3 Anderson et al, PRC 9 ASD children in total. 6 delayed children in total. Still colour photographs ASL Model 504 eye-tracking * Baseline: /
2006, Visual Mean age (months): 49.6
l Mean age (months): 46.3 presented centrally. system (Applied Science * Amplitude: significant differences between the ASD group
na
Scanning and 8M/1F 6M/0F Categories: children’s faces, Laboratory [ASL], 2001) with a and the delayed and TD groups. The ASD group showed
Pupillary Responses animal faces, toys, and Flock of Birds magnetic head pupillary constriction to internal features of the children’s face;
in Young Children Diagnosis: previous diagnosis of AD 9 TD children in total. landscapes. tracker (Ascension Technology pupillary dilation was observed in the delayed and TD groups;
with Autism (n=7) or PDD-NOS (n=2) confirmed Mean age (months): 49.8 Corporation, 1999) in a the latter two did not differ from each other.
Spectrum Disorder39 through administration of the 8M/1F partitioned interior room that * Latency: /
ADOS-G. was divided into participant
ur

and experimenter areas.

Gaze Tracker™ interface
program (ERICA, 2001)
4 Falck-Ytter, 2008, PRC 15 ASD children in total (7 autistic 15 TD children in total. 24 short videos (3.8s) showing Tobii 1750 and Tobii * Baseline: /
Face Inversion syndrome, 1 Asperger’s syndrome, Mean age (years): 4.11 (38d) facial expressions on a female Technology (Stockholm, * Amplitude: The mean pupil dilation to upright faces for ASD
Jo

Effects in Autism: A 7 PDD-NOS). 11M/4F model: angry, happy, fear, Sweden). and TD children did not differ, but the ASD group showed more
Combined Looking Mean age (years): 5.2 (11m) disgust, neutral, unlabelled dilation than the typically developing children for inverted
time and Range (years): 3 – 6 grimace. All faces were shown Near-infrared light. faces.
Pupillometric 12M/3F upright and inverted. First 12 * Latency: /
Study68 videos static representations of 17 inch TFT monitor.
Diagnosis: ADI-R, ADOS-G, RRBI the maximum of the
expression. Following 12
videos were dynamic
presentations.
5 Anderson et al, PRC Data obtained from Anderson et 6 mental age match children in Cfr. Anderson et al., 2006 Cfr. Anderson et al., 2006 * Baseline: Significantly larger in ASD than in MA and CA.
2009, Larger Tonic al., 2006 total. * Amplitude: /

f
Pupil Size in Young * Latency: /

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children With 9 chronological age match in
Autism Spectrum total.
Disorder40
6 Fan et al, 2009, PLR 24 children in total (autistic 44 typically developing Separate stimulation per eye Binocular pupillogram * Baseline: Not significantly different, but larger variation in the
Abnormal Transient disorder: 10, Asperger’s syndrome: children in total. under both light-adapted and recording system ASD group under both light and dark adaptations.
Pupillary Light Reflex 10, PDD-NOS: 4). Mean age (years) 10.4 ± 2.7 dark-adapted conditions * Amplitude: ASD group exhibited significantly smaller relative

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in Individuals with Mean age (years): 12.9 ± 4.3 Range (years): 6 – 16 (green led) Green LED at 530 nm (LXK2- constriction at DA 794.
Autism Spectrum Range (years): 7 – 20 23F/21M PM12-R00, Philips Lumileds * Latency: young individuals with ASD have a significantly
Disorders42 22M/2F Lighting) prolonged PLR latency (all light conditions).

7 Martineau et al, PRC
2011, Can pupil size
and pupil responses
during visual
Diagnosis: ADI-R and anamnesis
19 children with ASD in total.
Mean age (months): 118
Range (months): 41 – 181
16M/3F
e-
38 normally developing
children in total.
(19 children chronological age-
matched group.
Still colour photographs
(neutral faces, virtual faces and
a series of objects)
Infrared LED
FaceLAB monitoring system for
head position and orientation,
eye direction and eye-tracking
in real time.
* Baseline: The ASD group had a significantly smaller static
baseline pupil size, than either the MA-matched or CA-matched
groups. Significant difference in mean pupil size recorded
during the presentation of all the stimuli (4s), ASD had

Pr
scanning contribute Mean age {months}: 116 significantly smaller pupil size than both groups. In both cases
to the diagnosis of Diagnosis: ADI-R and anamnesis Range {months}: 44 –178 Gaze tracker software. little difference between the two groups. Pupil size was greater
autism spectrum 11M/8F, when the stimuli were faces, compared to objects. The same
disorder in And 19 children mental age- Two cameras. Gaze direction is difference was not observed when the stimuli were avatars.
children?65 matched group. monitored by the software * Amplitude: /
Mean age {months}: 87 maintained by the camera * Latency: /
Range {months}: 41 –136 system.
l 12M/7F)
na
Infrared diodes for pupil-
illumination.

8 Sepeta et al, 2012, PRC 20 ASD children in total. 18 TD children in total. Static images of emotional Tobii 1750 Eye-Tracking * Baseline: /
Abnormal social Mean age (years): 12.4 (2.5) Mean age (years): 13.7 (2.7) faces from the NimStim Face Technology, using Clearview * Amplitude: Children with ASD and TD controls showed similar
reward processing in Range (years): 8 – 18
ur

Range (years): 8 – 18 Stimulus Set. 20 young adult 2.2.0 software. pupillary responses; however, they differed significantly in their
autism as indexed 19M/1F 17M/1F individuals, each displaying sensitivity to gaze direction for happy faces. Specifically,
by pupillary four emotional expressions pupillary diameter increased among TD children when viewing
responses to happy Diagnosis: conformation with (angry, afraid, happy or happy faces with direct gaze as compared to those with averted
faces74 ADOS, WISC-III, WASI neutral). Gaze-direct condition gaze, whereas children with ASD did not show such sensitivity
Jo

and gaze-averted condition. To to gaze direction. There was no effect of gaze direction on pupil
control the fixation position, diameter for negative affect of neutral faces among either the
two fixation crosses before TD or ASD group.
each trial were shown. * Latency: /

9 Anderson et al, PLR STUDY 1: 12 ASD children in total STUDY 1: 9 DS (Down Blank grey slide (3.01x) for 3 Applied Science Laboratory * Baseline: ASD group had a larger adjusted mean tonic pupil
2013, Pupil and (8 AD, 4 PDD-NOS). Syndrome) children in total. min to collect tonic pupil size. (ASL) size that varied significantly from the adjusted means of the DS
Salivary Indicators of Mean age (months): 50.25 Mean age (months): 48.67 E6 eye-tracking system, Model and TD groups (whose tonic pupil size did not differ
Autonomic Range (months): 30 – 69 Range (months): 20 – 73 504 (ASL, 2008) with the significantly). We also found a strong correlation among tonic
 dysfunction in 11M/1F 7M/2F GazeTracker interface program pupil size and total ADOS-G scores. This demonstrate that the
Autism Spectrum STUDY 1: 11 TD children in (Eye-Gaze Response Interface larger tonic pupil size in ASD is independent of CA.

f
Disorder57 Diagnosis: confirmed by ADOS-G. total. Computer Aid, 2001) setup in a * Amplitude: /

STUDY 2: 18 ASD children in total
(11 AD, 7 PDD-NOS).
Mean age (months): 57.78
Mean age (months): 51.73
Range (months): 34 – 69
10M/1F
STUDY 2: 19 TD children in
total.
Mean age (months): 52.26
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darkened interior room. * Latency: /
Cfr study 1 Cfr study 1. * Baseline: Significant between-group difference in tonic pupil
size, with the ASD group having a significantly larger adjusted
Video head tracker. mean tonic pupil size than the TD group. Significant correlation

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Range (months): 39 – 73 Range (months): 33 – 79 among tonic pupil size and ADOS-G total scores.
* Amplitude: /
Diagnosis: confirmed by ADOS-G. * Latency: /
10 Daluwatte et al, PLR 152 children with ASD in total (86 107 TD children in total. Cfr Fan et al. Other light Cfr Fan et al. * Baseline: /

e-
2013, Atypical classic autism, 32 Asperger’s Mean age (years): 10.9 ± 2.9 intensities. * Amplitude: ASD and NDD children have less relative
pupillary light reflex Syndrome, 34 PDD-NOS). Range (years): 6 – 17 constriction amplitude than TD children for all testing
and heart rate Mean age (years): 10.7 ± 3.4 79M/28F conditions.
variability in Range (years): 5 – 19 * Latency: ASD and NDD groups had a significantly longer
children with autism 135M/17F 36 children with latency than those of the TD group for all testing conditions. A

Pr
spectrum disorder60 neurodevelopmental disorders significant age effect on PLR latency was observed in the TD
Diagnosis: DSM-IV, ADOS, ADI-R, in total (7 Down syndrome, 5 group, but not in the ASD group.
evaluation by a paediatrician and Fragile X syndrome, 1
or neuropsychologist. Neurofibromatosis Type one, 1 The PLR parameters were significantly different between the
Prader-Willi, 22 idiopathic TD and ASD groups, and between the TD and NDD groups, but
intellectual impairment). not between the ASD and NDD groups.
Mean age (years): 9.9 ± 3.0
l Range (years): 5 – 17
na
27M/9F
11 Blaser et al, 2014, PRC 17 children with ASD in total (15 17 age-matched TD children in Visual search displays Tobii T120 eye tracker * Baseline: Significantly higher tonic pupil level in the ASD
Pupillometry children met stricter criteria for total. group as compared to the TD group, albeit a more modest one.
Reveals a autism). Mean age (months): 29.5 ± 2.6 * Amplitude: ASD group has greater phasic pupil response
Mechanism for the Mean age (months): 29.6 ± 4.8 Range (months): 25 – 34 during visual search. Lager phasic pupil response is associated
Autism Spectrum Range (months): 21 – 35
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7M/10F with better search performance, and is sufficient to account for

Disorder (ASD) 14M/3F search data.
Advantage in Visual * Latency: /
Tasks51 Diagnosis: ADOS
12 Nuske et al, 2014 PRC 21 children with ASD in total (15 21 TD children in total. Videos of people initially Tobii 120 binocular eye tracker * Baseline: No group differences were found
Jo

(a), Reactivity to
fearful expressions
of familiar and
unfamiliar people in
children with
autism: an eye-
tracking
pupillometry study38
13 Nuske et al, 2014 PLR
(b), Brief report:
Autistic Disorder, 6 ASD).
Mean age (years): 3.98 (1.05)
Range (years): 2 – 5
18M/3F
Diagnosis: ADOS
26 children with ASD in total (17
Autism, 8 ASD).
Mean age (years): 4.27 (0.60)
Range (years): 2 – 5
18M/3F
21 TD children in total.
Mean age (years): 4.27 (0.60)
showing a neutral expression,
which gradually changed into a
prototypic fear expression. Ten
of the videos featured people
familiar to the children (ASD:
intervention therapists and
child-care teachers), and 10
featured unfamiliar people.
Six 50 % grey slides
(luminosity: 128; mean screen
and Tobii Studio software
(versions 3.2.3 Tobii,
Stockholm, Sweden).
Photometer (model PLMX,
Quantam Instruments, Barlett,
IL, USA).
Tobii 120 binocular eye tracker
and Tobii Studio software
* Amplitude: No difference between the groups on peak
amplitude to familiar, but the ASD group had reduced peak
amplitude to unfamiliar fear, compared to the TD group, who
had a greater peak amplitude to unfamiliar fear than to familiar
fear; the ASD group did not differentiate between familiarity
conditions.
* Latency: children with ASD showed a longer response latency,
compared to the TD group, across the familiarity conditions.
* Baseline: The ASD group did not differ from the typically
developing group on baseline pupil diameter.
 Evidence for
Normative Resting-
State Physiology in
Autism109
14 Nuske et al, 2014 PRC
(c), Pupillometry
reveals reduced
unconscious
emotional reactivity
in autism36
15 Daluwatte et al, PLR
2015, Association
between pupillary
light reflex and
sensory behaviours
in children with
autism spectrum
disorders58
16 Krach et al, 2015, PRC
Evidence From
Pupillometry and
fMRI Indicates
ur
Reduced Neural
Response During
Vicarious Social Pain
but Not Physical
Jo
Pain in Autism76
17 Nuske et al, 2015, PRC
No Evidence of
Emotional
Dysregulation or
Aversion to Mutual
Mean age (years): 4.02 (0.99)
21M/4F
Diagnosis: DSM-IV-TR criteria,
confirmation with ADOS
19 children with ASD in total (12
AD, 7 ASD).
Mean age (years): 3.97 (1.06)
Range (years): 2 – 5
17M/2F
Diagnosis: ADOS
Daluwatte et al., 2013
l also had smaller PLR constriction amplitudes.
na
16 ASD patients in total (1 autism,
14 Asperger’s syndrome, 1 atypical
autism).
Mean age (years): 21.50 ± 2.9
Older than 18 years
16M/0F
Diagnosis: ICD-10, DSM-IV, ADOS
Pupil measurements were available
for eleven ASD patients.
23 children with ASD in total (15
AD, 8 ASD).
Mean age (years): 4.05 (1.01)
Range (years): 2 – 5
19M,4F
18M/3F
19 TD children in total.
Mean age (years): 4.20 (0.80)
Range (years): 2 – 5
16M/3F
e-
Pr
Daluwatte et al., 2013
16 healthy controls in total.
Mean age (years): 24.31 ± 2.9
Older than 18 years
16M/0F
Matched for sex, age, and
verbal IQ (Wechsler).
Pupil measurements were
available for eleven healthy
control patients.
21 TD children in total.
Mean age (years): 4.27 (0.60)
Range (years): 2 – 5
18M, 3F
illuminance: 8.42 lx), and were
shown on the eye-tracking
monitor. Three durations were
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used twice, 0.5, 1 and 2 s each
shown after a scrambled image
(1 s) that was matched on
luminosity to the grey slides.
Forward and backward
pr
masking paradigm, fearful and
neutral facial expressions were
presented for three different
exposure conditions: 30ms,
300ms, and 2 s1. Scrambled
face image as mask stimulus.
Daluwatte et al., 2013
PP (physical pain): 56 digital
colour photographs of a
person in painful or non-
painful situations. Participants
needed to estimate the
intensity of physical pain.
SP (social pain): 50 sketches of
a protagonist in either socially
undesirable or neutral public
scenarios. Participants needed
to estimate the
embarrassment.
Continuous videos were taken
of adults, first looking out to
the side of the camera (for the
averted gaze condition) and
then looking directly at the
(versions 3.2.3 Tobii,
Stockholm, Sweden).
f
Photometer (model PLMX,
Quantam Instruments, Barlett,
IL, USA).
Tobii 120 binocular eye tracker
and Tobii Studio software
(versions 3.2.3 Tobii,
Stockholm, Sweden).
Photometer (model PLMX,
Quantam Instruments, Barlett,
IL, USA).
Daluwatte et al., 2013
MRI-compatible Eyelink-1000
device during both fMRI
paradigms.
Tobii 120 binocular eye tracker
and Tobii Studio software
(versions 3.2.3 Tobii,
Stockholm, Sweden).
* Amplitude: /
* Latency: /
* Baseline: /
* Amplitude: The TD group responded more to the fear
expressions then neutral expressions in the 30ms and 300ms
conditions, the ASD group showed the opposite pattern,
responding more to the neutral faces than the fearful faces.
The groups did not differ on the 2s condition. For the TD group,
peak amplitude was not different across the exposure
conditions. For the ASD group, peak amplitude in the 30ms was
significantly different to the 2s condition, and the 300ms was
marginally different from the 2s condition. There was no
difference between the 30ms and 300ms condition.
* Latency: /
* Baseline:
* Amplitude: PLR constriction amplitude correlated with total
sensory score in all light-adapted tests in ASD group. This
correlation was not observed in TD children. ASD children with
more atypical sensory behaviours (i.e. lower sensory scores)
* Latency: /
* Baseline: /
* Amplitude: There was a significant main effect for increase in
pupil diameter during PP compared to PN in both groups and
no significant effects of Group or Group x Condition interaction.
The SP task revealed a very different picture. The groups
differed markedly on all neurobiological parameters. Greater
pupil dilation during SP in the HC group which was not evident
in ASD individuals.
* Latency: ?
* Baseline: /
* Amplitude: both groups dilated their pupils more to mutual
than averted eye gaze, across the familiarity conditions. The
ASD group showed more pupil dilation to unfamiliar compared
to familiar faces. The TD group did not show this difference.
 Gaze is Preschoolers camera (for the mutual gaze Photometer (model PLMX, * Latency: /
with Autism Diagnosis: ADOS condition), and vice versa. Quantam Instruments, Barlett,

f
Spectrum Disorder: 6 of the videos featured people IL, USA).

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An Eye-Tracking familiar to the children (ASD:
Pupillometry Study66 intervention therapists and
child-care teachers), and 6
featured unfamiliar people.
Scrambled images as buffer for
the pupillary light reflex.

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18 * Nyström et al, PLR 29 children at risk in total. 15 TD children in total. Each stimulus 6s, small central Tobii 1750 eye tracker (Tobii * Baseline: No group differences.
2015, Mean age (days): 312 (15) Mean age (days): 313 (14) fixation point on a black Technology, * Amplitude: Infants at risk had larger constriction amplitudes
Hypersensitive 13M/16F 8M/7F background (0.9 lux) that Danderyd, Sweden). (relative constriction).
pupillary light reflex flashed white (190 lux) for * Latency: Infants at risk had shorter latency (TD significantly

e-
in infants at risk for 75ms. larger).
autism34
19 Richey et al, 2015, PRC 15 ASD adults in total. 15 NT controls in total. Neutral closed-mouth faces, Psychology Software Tools Inc., * Baseline: /
Neural Mechanisms Mean age (years): 26.1 (8.1) Mean age (years): 27.4 (8.3) followed by cognitive (Pittsburgh, PA, USA) * Amplitude: both groups were characterized by relatively
of Emotion 13M/2F 13M/2F reappraisal instruction (16 equivalent increases in pupil diameter during both regulation

Regulation in Autism
Spectrum Disorder80
Pr
think positive/ 16 think fMRI via an strategies.
Diagnosis: ADOS-G, WASI, Social negative). MR-compatible miniature * Latency: /
Responsiveness Scale, RBS-R analog video camera
(Resonance Technology, Inc.)

Viewpoint software (Arrington

Research)
20 Aldaqre et al, 2016, PRC l
18 adults with ASD in total (15 18 NT adults in total. Modified version of Tobii T60 eye-tracker (Tobii * Baseline:
na
Sensitivity to Asperger syndrome, 3 childhood Mean age (years): 36.9 (13.9) Posner’s spatial cueing Technology, Sweden) and the * Amplitude: overall increase in pupil dilation for the
communicative and autism). 9M/9F paradigm. software Tobii Studio (version incongruent cue condition compared to the congruent cue
non‑ communicative Mean age (years): 37.8 (10.8) 3.2.2, Tobii Technology, condition. Increased mental effort in the incongruent condition
gestures in 9M/9F Additionally: 14 TD adolescents Sweden). is reflected by this increase in pupil dilation, and it can be
adolescents and in total. attributed to participants having to reallocate their attention to
adults with autism Additionally: 14 adolescents with
ur

Mean age (years): 14.3 (1.1) target location after the target has already appeared.
spectrum disorder: ASD in total (11 Asperger 9M/5F Participants had overall larger pupils for the pointing than the
saccadic and syndrome, 3 childhood autism). grasping cues. Additionally, pupil diameter increased as SOAs
pupillary Mean age (years): 16.4 (1.9) got longer, potentially indication that pupil size dilated
responses67 10M/4F gradually over time.
Jo

The difference in pupil dilation between pointing and grasping

Diagnosis: ICD-10 criteria, AQ, SRS cues was particularly present in the ASD group.
* Latency: /
21 Kang et al 2016, PLR 34 ASD children in total. 33 TD children in total. Large black and white circles, LCD monitor (Vizio SV420M). * Baseline: children with ASD had significantly larger average
Evaluation of the Mean age (years): 5.79 Mean age (years): 6.06 presented on a uniform grey baseline pupil size than controls.
Pupillary Light Reflex 28M/6F 21M/12F background, and alternating Spectroradiometer (PR 650) * Amplitude
As a Potential every second. * Latency: Children with ASD had significantly longer mean PLR
Biomarker in Infrared-based eye-tracker latency than controls.
Children with (Tobii X120).
 Autism Spectrum Children with ASD had marginally significantly longer mean
Disorder35 dilation time than controls.

f
22 Müller et al, 2016, PRC 33 ASD adolescents in total. 23 TD adolescents in total (age- MASC consists of a 15-min film Tobii TX-300 eye-tracker (Tobii * Baseline: /

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Validation of the Mean age (years): 15.6 (1.9) matched. clip depicting four interacting AB, Stockholm) with Tobii * Amplitude: The ASD group exhibited a significantly reduced
Movie for the Range (years): 11.5 – 19.5 Mean age (years): 16.3 (2.4) characters (2 females, 2 males) Studio 3.2 software. mean pupil dilation during the MASC, but not before the MASC
Assessment of Social 27M/6F Range (years): 11.9 – 19.7 having dinner together. The on the first introductory slide.
Cognition in 14M/9F film is paused and the I-VT fixation filter. * Latency: /
Adolescents with 6 ASD participants were excluded participants are asked
ASD: Fixation from all eye-tracking analyses. 3 TD adolescents were questions.

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Duration and Pupil excluded from all eye-tracking
dilation as Diagnosis: ADOS-G, ADI-R and analyses.
Predictors of clinical evaluation
Performance71

e-
23 Nuske et al, 2016, PRC 20 children in total (14 children 20 children in total. Videos of an actor opening a Tobii 120 binocular eye tracker * Baseline: /
Others’ emotions autistic disorder, 6 children ASD). Mean age (years): M (SD) 4.25 plain box and reacting and Tobii Studio software * Amplitude: difference pre- to post-box in the TD group only,
teach, but nog in Mean age (years): M (SD) 4.03 (0.63) emotionally to the occluded ASD no difference.
autism: an eye- (1.09) Range: 24-60 months object inside (happy condition Social-emotional calibration is diminished in children with
tracking Range: 24-60 months 16M/4F and fear condition). An up- autism.

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pupillometry study37 16M/4F close image of the box was More social-emotional calibration on the happy condition was
shown before and after the associated with less severe autism symptoms.
Diagnosis: ADOS scene (measurement pupil * Latency: /
diameter).
24 * Wagner et al, PRC 29 high-risk infants for ASD in total. 20 low-risk controls in total. Each infant saw colour images Tobii T60 monitor using * Baseline: /
2016, Greater Pupil Mean age (days): 282 (10) Mean age (days): 280 (9) of one of the four female faces Clearview software (Tobii * Amplitude: HRA- showed larger pupil size than LRC in
size in Response to 17M/12F 13M/7F expressing three different Technology AB) response to emotional faces
Emotional Faces as l emotions (fearful, happy and * Latency: /
na
an Early Marker of High risk for ASD = having an older neutral).
Social- sibling diagnosed with ASD. One important limitation of the present work is that there was
Communicative Diagnosis: ADOS no consistent baseline period administered during the eye-
Difficulties in Infants tracking session and therefore very little pre-task pupil data
at High Risk for was available
Autism56
ur

25 DiCriscio et al, 2017, PRC 12 children with ASD in total (4 30 NT children in total. Black screen with a grey square Tobii studio. * Baseline: we do not find that baseline pupil diameter is linked
Pupil adaptation comorbid diagnosis of at least one (12) and a white screen with a with autism traits in the current study. Older age results in
corresponds to of the following: ADHD, learning grey square (12). Each stimulus Unknown eye tracker. smaller baseline pupil size.
quantitative disorder, language disorder, and (Total ASD/non-ASD: 42 was displayed for 5 seconds * Amplitude: Amplitude of dilation of the pupil in the light
Jo

measures of autism
traits in children69
mild to moderate intellectual
disability).
Identifying participants based on
patient referral, from health
system wide advertisement and
the surrounding community
Mean age (years): 8.95 ± 2.59
Range (years): 5-16
21M21F)
before switching to the condition was found to be significantly related to SRS Total T-
alternate stimulus display. The score, as well as all SRS subscales with the exception of Social
task began with the Motivation and RBRI. Individuals carrying an ASD diagnosis also
presentation of a grey screen. had significantly reduced dilation amplitude relative to the non-
ASD group.
Latency: latency to dilate is larger when older, higher SRS
correlates with smaller latency to constrict (no relationship
with diagnosis).
26 Dinalankara et al, PLR 52 children in total. 52 children in total. White projector screen Remote PLR instrument (rPLR) * Baseline: base pupil size increased with age in the TD group,
2017, Atypical Range: 24-72 months Range: 24-72 months illuminated for 100ms using a but not in the ASD group. The two different age trends of

f
Pupillary Light Reflex (24 months: 10, (24 months: 11, green LED (530 nm). Subject’s pupil illuminated resting pupil size in the ASD and TD groups appeared to

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in 2-6-Year-Old 36 months: 11, 36 months: 9, using an 850-nm near infrared intersect at 4 years old. Children with ASD younger than age 4
Children With 48 months: 10, 48 months: 11, (NIR) LED array. may have a larger pupil, while children with ASD older than age
Autism Spectrum 60 months: 10 60 months: 10 4 could have a smaller pupil than TD children.
Disorders44 72 months: 11) 72 months: 11) Projector screen (152.4 cm x * Amplitude: /
121.9 cm) * Latency: prolonged PLR latency present in 2-6-year-old
Diagnosis: DSM-IV criteria at the children with ASD, but less difference at 2 years old

pr
Thompson center. Confirmed using (explanation for Nyström?).
ADOS/paediatric autism specialist.

27 Lawson et al, 2017, PRC 24 ASD adults in total (19 Asperger, 25 TD adults in total. Auditory cues were either 330- Auditory cues generated in * Baseline: The results demonstrated no group differences in

Adults with autism
overestimate the
volatility of the
sensory
environment49
3 autistic disorder, 1 high-function
autism, 1 atypical autism).
Mean age (years): 35,5
Range (years): 20 – 61
18M/6F
e-
Mean age (years): 36
Range (years): 19 – 62
ADDITIONAL: 57 NT volunteers
in total.
Hz or 660-Hz pure tones.
Stimuli were grayscale and
comprised six different face
identities (three male and
MATLAB R2012b (Mathworks)
and presented with the Cogent
toolbox, via Sennheiser HD 201
headphones.
tonic pupil size in this sample.
* Amplitude: The phasic pupil response to surprising outcomes
revealed a significant increase in pupil size in NT participants,
the ASD group did not show this distinction between UE and E
trials.

Pr
Mean age (years): 27.1 female) or six different images Infrared eye tracker * Latency: /
Diagnosis: DSM-IV or ICD-10 Range (years): 19 – 50 of houses, masked by an (Cambridge Research Systems).
criteria, WAIS, ADOS for 25M/32F ellipse (NimStim Face Identity
confirmation Set). Gaussian noise was added
Eye-tracking data are available to outcome images.
Eye-tracking data are available for for only 14 subject with ASD.
only 11 subject with ASD.
28 Garon et al, 2018, PRC 30 ASD adults in total. l 59 TD adults in total. SoMoral task (16 moral Tobii T60XL eye-tracker during * Baseline: /
na
Visual Encoding of Mean age (years): 24.41 (24.71) Mean age (years): 22.80 (6.39) dilemmas). the SoMoral task. Tobii 24-inch * Amplitude: pupil dilation was similar across groups.
Social Cues Range (years): 17 – 34 Range (years): 16 – 44 screen. * Latency: /
Contributes to 23M/7F 33M/26F
Moral Reasoning in Tobii Studio 3.2 eye-tracking
Autism Spectrum Diagnosis: ADI-R, ADOS-G, DSM-IV software.
Disorder: An Eye-
ur

Tracking Study77
29 Gotham et al, 2018, PRC
Pupil response to
social-emotional
Jo
21 adults with ASD in total. 13 TD-depressed adults in Passive-viewing task in which Spectral Visual Saliency * Baseline: /
Mean age (years): 22.4 (4.6) total. emotionally-salient faces Toolbox Matlab. * Amplitude: Responsive pupil dilation in the ASD cohort
Range (years): 18 – 33 Mean age (years): 23.9 (3.3) (happy, sad, angry and neutral) tended to be significantly lower than TD-depressed initially but

material is 19%F Range (years): 19 – 32 were briefly presented for 400 increased to comparable levels by trial end. When viewing sad
associated with 54%F milliseconds, then followed by faces, individuals with ASD who had higher depression scores
rumination and Diagnosis: ADOS-2, SRS-2, AQ, a mask for 8 seconds. resembled TD-depressed participants’ faster, larger and
depressive SCID-5, MINI 5.0, BDI-II, RRS, RBS-R 19 TD-controls adults in total. A fixation cross was first briefly sustained pupil response. Within ASD, depressive symptoms
symptoms in adults Mean age (years): 26.6 (4.5) displayed. uniquely predicted early pupil response to sad faces, while
with autism Range (years): 21 – 35 rumination and depression scores each independently
spectrum disorder78 56%F predicted sustained pupil response.
* Latency: /
30 Laeng et al, 2018, PRC 11 ASD participants in total. 24 controls in total. Asahi brightness illusion and SMI software. * Baseline: The two groups did not differ in average pupil size at
Pupillary responses Mean age (years): 37.8 (4.01) Mean age (years): 29.9 (2.4) control pictures. Three baseline (ASD = 11.3; SE=0.5; Controls=12.3; SE=0.4).
 to Illusions of
Brightness in Autism
Spectrum Disorder62
31 Lynch et al, 2018, PLR
Pupillary Response
and Phenotype in
ASD: Latency to
constriction
discriminates ASD
from Typically
Developing
Adolescents41
32 Nyström et al, 2018, PLR
Enhanced pupillary
light reflex in infancy
is associated with
autism diagnosis in
toddlerhood47
33 Aguillon-Hernandez PRC
et al, 2019, The
pupil: a window on
social automatic
processing in autism
spectrum disorder
ur
children43
Jo
Range (years): 23 – 65
8M/3F
Diagnosis: ICD-10
10 adolescents with ASD in total.
Mean age (years): 16.15
Range (years): 13.11 – 17.2
8M/2F
Diagnosis: DSM-V criteria (ASD
level 1 or level 2), confirmed with
ADI-R
29 HR-ASD (high risk infants later
diagnosed with ASD) infants in
total.
Mean age (months): 9.6 (31.65d)
22M/7F
Diagnosis: ADI-R, ADOS-2, Vineland
Adaptive Behaviour Scales and
MSEL.
l
na
STUDY 1: 22 ASD children in total.
Mean age (years): 9 ± 4
Range (years): 4 – 16
18M/4F
Diagnosis: ?
STUDY 2: 56 ASD children in total.
Mean age (years): 7 ± 3
Range (years): 3 – 12
50M/6F
Range (years): 23 – 66
10M/14F
12 TD adults in total.
Mean age (years): 15.20
Range (years): 11.5 – 17.8
7M/5F
e-
118 HR-no-ASD infants in total.
Mean age (months): 9.43
Pr
(26.80d)
56M/62F
40 TD infants in total.
Mean age (months): 9.8
(25.40d)
20M/20F
STUDY 1: 47 TD children in
total.
Mean age (years): 10 ± 4
Range (years): 3 – 16
25M/22F
22 in the TDsg group to match
in age and gender.
Mean age (years): 9 ± 4
Range (years): 3 – 16
18M/4F
STUDY 2: 61 TD children in
total.
Mean age (years): 7 ± 3
Range (years): 3 – 12
36M/25F
coloured patterns (yellow,
purple, or green) were
presented during the test.
oo
The participants also
completed a task.
Cfr. Daluwatte et al., 2013
pr
Cfr Nyström et al., 2015
Colour photographs of five
objects, five virtual faces and
five real faces (neutral, happy
or sad).
Face photographs with a
neutral expression and face
videos with a dynamic facial
expression (neutral, happy or
sad).
SMI RED500 remote eye-
tracking device by
f
SensoMotoric Instruments.
Face-LAB5VC eye-tracking
technology (Seeing Machines,
2013).
100 lumen LED ADC Adlite Pro
standard medical penlight.
Cfr Nyström et al., 2015
FaceLAB head-free binocular
eye-tracking system.
GazeTracker software.
FaceLAB head-free binocular
eye-tracking system.
GazeTracker software.
* Amplitude: The effect sizes of the responses of the ASD group
and Control group to the bright or the dark patterns appeared
to be similar.
* Latency: /
* Baseline: /
* Amplitude: /
* Latency: The ASD group showed a delay in pupil response.
Larger latency to max constriction in ASD, latency in return to
baseline.
* Baseline: No group differences.
* Amplitude: Main effect of group in terms of relative
constriction amplitude: HR-ASD significantly differs from HR-no-
ASD and TD at 10m.
* Latency: No group differences.
* Baseline: Basil pupil size was lower in ASD children compared
to TD children, but no difference was found between ASD and
TDsg children.
* Amplitude: The comparison of ASD and TD children in ERPD
revealed a marginal difference in the amplitude of the ERPD in
response to virtual faces whereas no difference was observed
for objects, neutral faces or emotional faces. No such
difference was found between ASD and TDsg children.
* Latency: /
* Baseline: basal pupil size measure in response to colour
interstimuli was comparable between ASD children and TD
children.
* Amplitude: the comparison between ASD and TD children
revealed a large significant difference in the amplitude of the
pupil dilation in response to dynamic neutral faces, whereas no
difference was observed for static faces.
* Latency: no latency difference was reported (?)
34 Bast et al, 2019, PRC 23 ASD adolescents in total (7 did 24 NTC adolescents in total. One PD progression was Eye tracker unknown. * Baseline: There were no group differences for baseline PD.
Pupil Dilation not meet ADOS autism spectrum Mean age (years): 15.8 (2.4) assessed during each stimulus * Amplitude: The ASD group exhibited increased constriction

f
Progression but ADI-R cut-off criteria and Range (years): 11.6 – 20.5 presentation (social stimuli, amplitude and increased dilation amplitude.

Modulates Aberrant
Social Cognition in
Autism Spectrum
Disorder53
clinical experts confirmed
diagnosis).
Mean age (years): 15.9 (2.8)
Range (years): 12.7 – 24.5
18M/5F
oo
19M/5F MASC). 43 video sequences * Latency: The ASD group exhibited increased dilation latency.
depicting four characters There were no group differences for constriction latency.
eating dinner together.
In ASD compared with NTC, a distinct PD progression was
Followed by a multiple-choice observed with increased constriction amplitude, increased
question presented on-screen dilation latency, and increased dilation amplitude that

pr
Diagnosis: ADOS, ADI-R, and clinical with white text on black correlated with PD progression components. These
evaluation, Wechsler Intelligence background regarding the components predicted social cognition performance. The first
Scale for Children or the Wechsler depicted characters’ cognition, and second PD components correlated positively with MASC
Adult Intelligence Scale. emotion, or intention. behavioural performance in ASD but negatively in NTC. These

e-
PD components may be interpreted as indicators of sensory-
perceptual processing and attention function.
35 Galazka et al, 2019, PRC 54 ASD individuals in total (24 63 age- and gender-matched Colour photographs of happy Tobii 120 eye tracker (Tobii, * Baseline: We found no effect of group on the baseline
Pupillary Contagion autistic disorder, 27 Asperger’s TD individuals in total. (20) and sad (20) faces (ten Stockholm, Sweden). measures of pupil size, nor any significant interaction with
in Autism73 syndrome, 2 PDD-NOS). Mean age (years): 20.56 (7.38) different female facial group as a factor.

Pr
Mean age (years): 21.37 (9.17) Range (years): 10 – 44 identities) created using * Amplitude: No group differences, and there were no
Range (years): 10 - 44 57M/6F Daz3Studio software. Different significant interactions with group as a factor.
49M/5F pupil sizes. * Latency: /

Diagnosis: DSM-IV, ADI-R, DISCD, Test images (fixation cross).

ADOS
36 Li et al, 2019, Third- PRC 20 ASD children in total. 19 TD children in total. Dynamic visual stimuli Tobii X60 eye tracker * Baseline: /
Party Sociomoral l
Mean age (years): 5.36 (1.08) Mean age (years): 5.28 (0.51) depicting intentional or (Tobiitech Technology, * Amplitude: No main effect of group. The pupil dilated more
na
Evaluation in Range (years): 4.00 – 7.10 Range (years): 4.67 – 6.25 accidental harm to persons or Stockholm, Sweden). when the victim was an object than when it was a person, and
Children With 17M/3F 16M/3F damage to object. this difference was greater for the ASD group than the TD
Autism Spectrum63 group.
Diagnosis: DSM-IV, confirmation Matched by age, IQ, and verbal * Latency: /
with AQ-Child. mental age.
37 Ring et al 2019, A PRC 27 ASD individuals in total.
ur

30 TD individuals in total. Words, pictures, shapes, and Tobii TX300 remote eye * Baseline: ASD mean baseline pupil 3.21 (0.57) and TD mean
Physiological Marker Mean age (years): 42.31 (11.5) Mean age (years): 43.98 (12.7) non-words presented in the tracking system. baseline pupil 3.17 (0.45)
of Recognition Range (years): 27 – 64 Range (years): 22 – 65 center of the screen on the * Amplitude: A pupil size ratio (pupil size for test item divided
Memory in Adults 23M/4F 23M/7F same grey background and Matlab routines. by baseline) for old (studied) and new (unstudied) materials
with Autism images were black and white. indicated larger pupils for old compared to new materials only
Jo

Spectrum disorder?
- The pupil Old/New
Effect52
38 Traynor et al, 2019, PRC
Eye Tracking Effort
Expenditure and
Autonomic Arousal
to Social and
Diagnosis: DSM-IV-TR, and 21
individuals confirmed with ADOS.
10 ASD adults in total.
Mean age (years): 25.4 (15.3)
Range (years): 16 – 34
8M/2F
Matched on VIQ, PIQ and FIQ,
chronological age and gender.
19 TD controls in total.
Mean age (years): 20.7 (2.42)
Range (years): 18 – 28
10M/9F
During baseline recording, a
fixation cross remained on the
screen.
Eyelink II head-mounted eye
tracking system (SR Research
Ltd.)
for the TD but not the ASD group. Pupil size in response to old
versus new items was positively related to recognition
accuracy, confirming that the pupil Old/New effect reflects a
memory phenomenon in the ASD group.
* Latency: /
* Baseline: ASD mean 882.319 (420.627) ASD range: 371.6660 –
1594.350. Control mean 631.835 (240.447) Control range:
337.340 – 1277.685.
* Amplitude: ASD subjects demonstrated larger pupil size to CI
images, compared to both social and neutral images, and
 Circumscribed Diagnosis: pre-existing diagnosis Images of people and objects Eyelink II system using Data importantly, no difference in pupil size when viewing social or
Interest Stimuli in given by a licensed practitioner. on the screen. 40 unsaturated, Viewer software (SR Research neutral images. The control group demonstrated no differences

f
Autism Spectrum WASI-II, AQ black-and-white images of Ltd.) in pupil size when viewing interest and social stimuli.

oo
Disorder79 each participant’s self- * Latency: /
identified interest, 40 social Experiment Builder (SR
images, and 40 images of Research Ltd.)
neutral objects.

The experiment consisted of a

pr
passive (60 images) and an
active block (60 images).
39 Boxhoorn et al, PRC 18 ASD children in total. 31 TD children in total. Participants completed a Tobii X2-30 binocular eye * Baseline: Table 2
2020, Pupil dilation Mean age (years): 13.8 (2.6) Mean age (years): 14.4 (2.7) visuospatial orienting task in a tracker (Tobii Technology AB, * Amplitude: Table 2

e-
during visuospatial Range (years): 8 – 18 Range (years): 8 – 18 quit and dimly lit testing room. Sweden). Stronger stimulus-evoked PD amplitude responses were
orienting 16M/2F 15M/16F observed in ASD (+ADHD) relative to TD and ADHD- following
differentiates nonspecific relative to specific cues.
between autism 14 ASD + ADHD children in total. 25 ADHD children in total. * Latency: Table 2
spectrum disorder Mean age (years): 12.6 (3.0) Mean age (years): 13.5 (2.4) Post hoc comparisons showed shorter cue-evoked PD latencies

Pr
and attention- Range (years): 8 – 18 Range (years): 8 – 18 in ADHD – relative to TD and ASD + ADHD. Cue-evoked PD
deficit/hyperactivity 12M/2F 25M/3F latencies did not differ between ASD- and TD, ASD + ADHD and
disorder81 TD.
Diagnosis: DSM-V, confirmation
with ADOS and ADI-R

40 Del Valle Rubido et PRC l

al, 2020, Exploring
Social Biomarkers in
High-Functioning
Adults with Autism
and Asperger’s
Versus Healthy
ur
38 ASD in total (high-functioning). 19 HC in total. Different videos in 7 different Tobii T60XL. * Baseline: /
na
Mean age (years): 24.2 (5.8) Mean age (years): 26.7 (4.3) paradigms. * Amplitude: The ASD group had larger pupil sizes than the HC
Range (years): 18 – 40 Range (years): 18 – 40 group during all seven eye tracking paradigms.
38M/0F 19M/0F * Latency: /
Diagnosis: DSM-IVTR, ADOS

Controls: A Cross-
Sectional Analysis54
41 Granovetter et al, PRC 23 ASD individuals in total. 24 age- and gender-matched Task stimuli (Psychophysics) Pupil area and coordinates * Baseline: There was no significant effect of group on the
2020, Mean age (years): 32.04 (8.12) controls in total. with-out and with were measured using Eyelink median baseline pupil size. Moreover, among participants with
Jo

Uncharacteristic
Task-Evoked
Pupillary Responses
Implicate Atypical
Locus Ceruleus
Activity in Autism50
Range (years): 21 – 49
8.70%F
Diagnosis: confirmed by an expert
clinician. ADOS, Wechsler
Abbreviated Scale of Intelligence
Mean age (years): 28.38 (6.74)
Range (years): 21 – 47
8.33%F
accompanying distractors.
Green circle at center. A set of
15 lowercase grey letters
randomly appeared one at a
time in the circle. Keyboard
press when a letter appeared.
Then same task but with
distractor auditory stimuli.
1000.
Pupillometry data were
processed using MATLAB 9.5.0
(Mathworks)
ASD, there was no significant effect of adrenergic-related
medications on median baseline pupil size. This indicates that
there were likely no systematic differences in pupil size.
* Amplitude: Individuals with ASD exhibited smaller task-
evoked pupil response amplitudes than did controls in the
presence but not absence of distractor stimuli. No interaction
effect between group and task condition on pupil amplitude
responses to misses. Group membership can be predicted from
the difference in pupil amplitude responses in the presence
versus absence of distractors, only during hits and FA’s (and not
 misses). Clinical metrics correlate with pupil response
amplitude to hits.

f
* Latency: /

42 Hepach et al, 2020, PRC
Prosocial attention
in children with and
without autism
spectrum disorder:
Dissociation
30 ASD children in total.
Mean age (months): 40.90 (9.78)
Range (months): 25 – 59
22M/8F
Diagnosis: confirmed using ADOS-
25 TD children in total.
Mean age (months): 41.80
(10.80)
Range (months): 24 – 61
20M/5F
oo
Two types of videos: One of a
male adult stacking items to
build a tower while he was
observed by a female adult
(social condition) and one
video of self-propelled items
Tobii model T120 (Tobii
Technology, Stockholm,
Sweden) with an integrated
computer screen. Tobii Studio
(Version 3.4.3; Tobii
Technology).
* Baseline: TD children showed similar levels of baseline pupil
diameter than children with ASD. In addition, children showed
similar levels of baseline pupil diameter in the social condition
compared to the non-social condition.
* Amplitude: TD children showed similar increase in pupil
dilation in response to seeing the objects drop compared to

pr
between 2, Social Communication being stacked without any children with ASD. For TD children, pupil dilation in response to
anticipatory gaze Questionnaire, Mullen Scales of person present (non-social seeing the adult not being helped did nog change with DQ,
and internal Early Learning condition). Followed by a drop whereas their pupil dilation in response to seeing the non-
arousal72 video. The last scene was the social condition remain unresolved did increase with DQ. We

e-
resolution video. found a different pattern for children with ASD. Here children’s
resolution measure of pupil dilation increased with DQ in the
After each video neutral social condition, but not in the non-social condition.
stimuli (images) were * Latency: /
presented.

43 * Kercher et al, PLR
2020, A longitudinal
study of pupillary
light reflex in 6- to
24- month children55
23 high risk children in total.
6-mo (6.3±0.8): 5F/6M
12-mo (12.2±1.1): 2F/3M
18-mo (17.6±0.6): 8F/12M
24-mo (23.4±0.5): 6F/8M
Consists of children who have at
l
least one sibling diagnosed with
na
ASD.
Pr
19 low risk children in total.
6-mo (6.0±0.7): 2F/3M
12-mo (11.3±1.3): 9F/10M
18-mo (17.4±00.5): 9F/10M
24-mo (23.7±0.5): 8F/8M
Cartoon videos. PLR was
elicited by flashing the
projection screen using a
ceiling-mount green LED.
PLR (rPLR) instrument.
* Baseline: The HR group appeared to have a larger pupil than
the LR group. In comparison with the LR group, the pupils in the
HR group were bigger before stimulation.
* Amplitude: The pupils in the HR group remained bigger at the
maximal constriction; but the relative constriction was smaller.
* Latency: All timing parameters were similar in the HR and LR
groups.

The final dataset consists of PLR

measures from nine children (6 in
HR and 3 in LR) who successfully
completed test at all four ages, 24
ur

children (9 in HR and 15 in LR) who

successfully completed tests at
three ages, and nine children (8 in
HR and 1 in LR) who only
Jo

completed tests at two or one age.

44 Reisinger et al, 2020, PRC 42 ASD individuals in total. 29 age-, gender-, and IQ- 12 coloured photographs of Tobii (Stockholm, Sweden) * Baseline: /
Atypical Social 83.33%M matched DD controls. adult human faces T120 infrared binocular eye * Amplitude: The DD participants exhibited a significant
Attention and Mean age (years): 12.33 (5.80) 89.65%M (females=males) from the tracker. reduction in pupil diameter during fearful faces in comparison
Emotional Face Range (years): 3 – 25 Mean age (years): 12.33 (5.80) NimStim Face Stimulus Set, to TD participants. The ASD participants exhibited a significant
Processing in Autism Range (years): 3 – 25 each showing a calm, happy or Photometer (Minolta, LS-100, increase in pupil diameter during happy faces in comparison to
Spectrum Disorder: Diagnosis: DSM-V, ADOS-2, SCQ fearful facial expression. Prior Osaka, Japan). TD participants.
Insight From Face 62 age-, gender-matched TD a scrambled version of the face * Latency: The ASD group on average exhibited a significant
Scanning and controls in total. image was presented. increase in pupil reactivity across the last nine intervals in
Pupillometry75 88.79% comparison to the DD group. The DD group had a significant
 Mean age (years): 12.33 (5.80) reduction in pupil diameter across the last five intervals in
Range (years): 3 – 25 comparison to the TD group.

f
oo
Table 1. Included articles. *Only included in qualitative analyses because participants were HR-infants.

pr
e-
l Pr
na
ur
Jo
 Study RISK OF BIAS APPLICABILITY CONCERNS
PATIENT INDEX REFERENCE FLOW AND PATIENT INDEX TEST REFERENCE
SELECTION TEST STANDARD TIMING SELECTION STANDARD

1. Rubin et al  ?     
2. Van Engeland et al ? ?  ?   
3. Anderson et al ? ?  ?   
4. Falck-Ytter et al ? ?   ?  
5. Anderson et al ? ?  ?   
6. Fan et al ? ?     
7. Martineau et al ? ? ? ?   
8. Sepeta et al  ?     
9. Anderson et al ? ?     
10. Daluwatte et al ? ? ? ?   
11. Blaser et al ? ? ? ?   
12. Nuske et al ? ? ? ?   

of
13. Nuske et al ? ? ? ? ?  
14. Nuske et al ? ? ?    
15. Daluwatte et al ? ? ? ?   

ro
16. Krach et al ? ? ? ? ?  
17. Nuske et al ? ?   ?  
18. Nyström et al ? ? ? ?   
19. Richey et al
20. Aldaqre et al
21. Kang et al
?
?
?
?
?
?
?
?
?

-p
?

?


?






   
re
22. Müller et al ? ? ?
23. Nuske et al ? ? ?    
24. Wagner et al ? ? ? ?   ?
25. DiCriscio et al ? ? ?    ?
lP

26. Dinalankara et al ? ?  ?   
27. Lawson et al ? ? ? ?  ? 
28. Garon et al ? ? ? ?   
29. Gotham et al ? ? ?    
na

30. Laeng et al ? ?   ?  
31. Lynch et al ? ?  ? ?  
32. Nyström et al ? ?     
33. Aguillon-Hernandez et al ? ? ? ? ?  ?
   
ur

34. Bast et al ? ? ?
35. Galazka et al ? ? ?    
36. Li et al ? ?  ? ?  
37. Ring et al ? ?     
Jo

38. Traynor et al ? ?  ?   ?
39. Boxhoorn et al ? ? ? ?   
40. Del Valle Rubido et al ? ? ? ?   
41. Granovetter et al ? ? ? ?   
42. Hepach et al ? ? ? ?   
43. Kercher et al ? ? ? ?   
44. Reisinger et al ? ? ? ? ?  
Low Risk High Risk ? Unclear Risk
Table 2. Risk of bias, number of study corresponds to table 1.
3(A) Baseline pupil size
Study Author, year Type Outcome ES Variance Weight 95% CI: lower and upper
boundaries
1 Rubin et al, 1961 PLR 1 -6,17319 2,567128 0,38954 -9,31356 -3,03283
5 Anderson et al, 2009 PRC 2 1,174332 0,362564 2,758131 -0,00585 2,354513
5 Anderson et al, 2009 PRC 3 1,362061 0,311944 3,205708 0,267364 2,456759
6 Fan et al, 2009 PLR 4 -0,51709 0,066918 14,94368 -1,02412 -0,01007
6 Fan et al, 2009 PLR 5 -0,25646 0,065413 15,28741 -0,75775 0,244835
7 Martineau et al, 2011 PRC 6 -2,57552 0,192543 5,193632 -3,43556 -1,71548
7 Martineau et al, 2011 PRC 7 -2,18999 0,168369 5,939332 -2,99423 -1,38575
9 Anderson et al, 2013 PLR 8 1,1013 0,200609 4,984822 0,223428 1,979172
9 Anderson et al, 2013 PLR 9 1,8346 0,15367 6,507437 1,066264 2,602936
10 Daluwatte et al, 2013 PLR 10 -0,10897 0,015948 62,7051 -0,35649 0,138546
10 Daluwatte et al, 2013 PLR 11 0,035869 0,015927 0,325644 -0,21149 0,283227
11 Blaser et al, 2014 PRC 12 0,25 0,118566 8,434109 -0,4249 0,924895

of
12 Nuske et al, 2014 PRC 13 0,243613 0,106044 0,316516 -0,39465 0,881875
13 Nuske et al, 2014 PLR 14 -0,07927 0,087687 11,40415 -0,65967 0,501126
21 Kang et al. 2016 PLR 15 0,691033 0,063278 15,80317 0,197991 1,184075

ro
23 Nuske et al, 2016 PRC 16 0,120714 0,100182 9,981818 -0,49966 0,741085
26 Dinalankara et al, 2017 PLR 17 0,050532 0,038474 25,9917 -0,33392 0,434981
26 Dinalankara et al, 2017 PLR 18 3,49601 0,481911 2,075071 2,135382 4,856639
26 Dinalankara et al, 2017 PLR 19 0,874254 0,221128 4,522263 -0,04742 1,79593
26
26
26
Dinalankara et al, 2017
Dinalankara et al, 2017
Dinalankara et al, 2017
PLR
PLR
PLR
20
21
22
-p
0,181023
-1,17299
-2,01172
0,191689
0,234398
0,273796
5,216775
4,266256
3,652359
-0,67711
-2,12192
-3,0373
1,039157
-0,22406
-0,98614
re
27 Lawson et al, 2017 PRC 23 0,145 0,16827 5,942823 -0,65901 0,949007
30 Laeng et al, 2018 PRC 24 -0,53378 0,136646 7,318174 -1,25831 0,190744
31 Lynch et al, 2018 PLR 25 -0,00916 0,183335 5,454489 -0,84839 0,830064
lP

32 Nyström et al, 2018 PLR 26 0,2655 0,059994 16,66846 -0,21457 0,745574

33 Aguillon-Hernandez et al, 2019 PRC 27 -0,6249 0,069561 14,37591 -1,14184 -0,10796
33 Aguillon-Hernandez et al, 2019 PRC 28 -0,2187 0,034455 29,02337 -0,58252 0,145113
37 Ring et al 2019 PRC 29 0,078392 0,070424 14,19965 -0,44174 0,598528
na

38 Traynor et al, 2019 PRC 30 0,802113 0,163724 6,107824 0,009041 1,595186

41 Granovetter et al, 2020 PRC 31 0,03 0,088943 11,24318 -0,55454 0,614536
42 Hepach et al, 2020 PRC 32 0,234414 0,087622 11,41271 -0,34576 0,814593
ur

3(B) Latency
Study Author, year Type Outcome ES Variance Weight 95% CI: lower and upper
boundaries
Jo

6 Fan et al, 2009 PLR 1 2,669022 0,118084 8,468529 1,9955 3,342545

6 Fan et al, 2009 PLR 2 0,930223 0,07138 14,00952 0,406569 1,453877
6 Fan et al, 2009 PLR 3 3,005698 0,132342 7,556178 2,292673 3,718724
10 Daluwatte et al, 2013 PLR 4 0,959908 0,017704 56,48584 0,699121 1,220696
10 Daluwatte et al, 2013 PLR 5 1,191392 0,018665 53,57643 0,923617 1,459166
10 Daluwatte et al, 2013 PLR 6 1,190617 0,018661 53,58666 0,922868 1,458366
10 Daluwatte et al, 2013 PLR 7 1,08162 0,018183 54,9957 0,817323 1,345917
12 Nuske et al, 2014 PRC 8 0,698629 0,101049 0,317881 0,075581 1,321677
12 Nuske et al, 2014 PRC 9 0,871783 0,104286 0,322933 0,238834 1,504731
21 Kang et al. 2016 PLR 10 0,622491 0,062607 15,97277 0,132073 1,112908
21 Kang et al. 2016 PLR 11 0,648749 0,104166 9,600021 0,016162 1,281336
 26 Dinalankara et al, 2017 PLR 12 0,606767 0,040232 24,8561 0,213634 0,9999
26 Dinalankara et al, 2017 PLR 13 0,704667 0,202732 4,932625 -0,17784 1,587171
26 Dinalankara et al, 2017 PLR 14 3,327516 0,478829 2,088427 1,971245 4,683787
26 Dinalankara et al, 2017 PLR 15 1,514972 0,245555 4,072402 0,543723 2,486222
26 Dinalankara et al, 2017 PLR 16 2,020264 0,302037 3,310856 0,94309 3,097438
26 Dinalankara et al, 2017 PLR 17 2,761184 0,355094 2,816155 1,593225 3,929144
31 Lynch et al, 2018 PLR 18 0,512079 0,189293 5,282816 -0,34067 1,364832
32 Nyström et al, 2018 PLR 19 0,113905 0,059577 16,78506 -0,3645 0,592308
39 Boxhoorn et al, 2020 PRC 20 0,08 0,087879 11,37929 -0,50103 0,66103

3(C) Amplitude
Study Author, year Type Outcome ES Variance Weight 95% CI: lower and upper
boundaries
1 Rubin, 1961 PLR 1 -1,15516 0,524132 1,907915 -2,57414 0,263826
1 Rubin, 1961 PLR 2 -1,34676 0,550765 1,815657 -2,80135 0,107824
1 Rubin, 1961 PLR 3 -2,43587 0,779636 1,28265 -4,16649 -0,70525

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1 Rubin, 1961 PLR 4 -2,52518 0,804251 1,243393 -4,28291 -0,76745
1 Rubin, 1961 PLR 5 -6,84641 3,054071 0,327432 -10,2717 -3,42113
1 Rubin, 1961 PLR 6 -3,75284 1,232433 0,811403 -5,92873 -1,57694
1 Rubin, 1961 PLR 7 -3,4251 1,10174 0,907655 -5,48239 -1,36781

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1 Rubin, 1961 PLR 8 -1,8434 0,638784 1,565474 -3,40991 -0,27689
1 Rubin, 1961 PLR 9 -1,93486 0,657982 1,519799 -3,52473 -0,34498
1 Rubin, 1961 PLR 10 -2,08517 0,691552 1,446022 -3,7151 -0,45524
3 Anderson et al, 2006 PRC 11 -0,36117 0,225846 4,427801 -1,29263 0,570282
3
3
3
4
Anderson et al, 2006
Anderson et al, 2006
Anderson et al, 2006
Falck-Ytter, 2008
PRC
PRC
PRC
PRC
12
13
14
15
0,039649
-0,76741
-1,8159
-0,8974
-p 0,222266
0,238581
0,31382
0,15198
4,499116
4,191446
3,186543
6,579805
-0,88439
-1,72477
-2,91389
-1,6615
0,963693
0,189945
-0,71792
-0,1333
6 Fan et al, 2009 PLR 16 -0,42528 0,066272 15,08928 -0,92985 0,079286
re
6 Fan et al, 2009 PLR 17 -0,0516 0,064942 15,39827 -0,55109 0,447877
6 Fan et al, 2009 PLR 18 -0,61314 0,067728 14,76495 -1,12322 -0,10305
8 Sepeta et al, 2012 PRC 19 0,152907 0,105863 9,446153 -0,48481 0,790625
8 Sepeta et al, 2012 PRC 20 0,360607 0,107267 9,322569 -0,28132 1,002538
lP

8 Sepeta et al, 2012 PRC 21 0,246981 0,106358 0,308634 -0,39223 0,886188

8 Sepeta et al, 2012 PRC 22 0,3003 0,106742 0,319338 -0,34006 0,940659
8 Sepeta et al, 2012 PRC 23 0,455482 0,108285 0,360975 -0,18949 1,100454
8 Sepeta et al, 2012 PRC 24 0,424489 0,107926 0,346445 -0,21941 1,068392
8 Sepeta et al, 2012 PRC 25 0,469105 0,108451 0,32446 -0,17636 1,11457
8 Sepeta et al, 2012 PRC 26 0,490545 0,108722 0,305708 -0,15573 1,136815
na

10 Daluwatte et al, 2013 PLR 27 -0,37543 0,016197 0,301829 -0,62487 -0,12599

10 Daluwatte et al, 2013 PLR 28 -0,56213 0,016535 0,303991 -0,81416 -0,3101
10 Daluwatte et al, 2013 PLR 29 -0,52569 0,016458 0,303643 -0,77714 -0,27424
10 Daluwatte et al, 2013 PLR 30 -0,42002 0,016265 61,4805 -0,66999 -0,17005
11 Blaser et al, 2014 PRC 31 0,47 0,120896 8,2716 -0,21149 1,151493
12 Nuske et al, 2014 PRC 32 -0,03779 0,095255 10,49813 -0,64271 0,567133
ur

12 Nuske et al, 2014 PRC 33 -0,75236 0,101977 9,806158 -1,37826 -0,12646

14 Nuske et al, 2014 PRC 34 1,379508 0,130303 7,67441 0,671996 2,087019
14 Nuske et al, 2014 PRC 35 1,059162 0,120024 8,331668 0,38013 1,738193
14 Nuske et al, 2014 PRC 36 -0,02925 0,105274 9,498984 -0,66519 0,606694
Jo

16 Krach et al, 2015 PRC 37 0,293166 0,183772 5,44154 -0,54706 1,13339

16 Krach et al, 2015 PRC 38 -0,11381 0,182113 5,491109 -0,95024 0,72261
16 Krach et al, 2015 PRC 39 -0,07258 0,181938 5,49638 -0,90861 0,763439
16 Krach et al, 2015 PRC 40 -0,86546 0,198842 5,029131 -1,73946 0,008533
17 Nuske et al, 2015 PRC 41 -0,4557 0,093457 10,7001 -1,05489 0,143488
17 Nuske et al, 2015 PRC 42 0,017572 0,091101 10,97685 -0,57401 0,609157
17 Nuske et al, 2015 PRC 43 -0,33992 0,09241 10,8213 -0,93574 0,255901
17 Nuske et al, 2015 PRC 44 0,311345 0,092199 10,84612 -0,28379 0,906484
23 Nuske et al, 2016 PRC 45 -0,6726 0,105655 9,464774 -1,30969 -0,03551
23 Nuske et al, 2016 PRC 46 -0,71001 0,106301 9,407212 -1,34905 -0,07097
25 DiCriscio et al, 2017 PRC 47 -1,2501 0,116667 8,571429 -1,91957 -0,58063
26 Dinalankara et al, 2017 PLR 48 -0,0968 0,038507 25,96958 -0,48141 0,287817
26 Dinalankara et al, 2017 PLR 49 -0,33258 0,193543 5,166822 -1,19485 0,529696
26 Dinalankara et al, 2017 PLR 50 -0,31816 0,204551 4,888761 -1,20461 0,568297
 26 Dinalankara et al, 2017 PLR 51 0,016012 0,190915 5,237928 -0,84039 0,872412
26 Dinalankara et al, 2017 PLR 52 -0,76707 0,21471 4,657443 -1,67528 0,141127
26 Dinalankara et al, 2017 PLR 53 -0,38461 0,18518 5,40015 -1,22804 0,458832
28 Garon et al, 2018 PRC 54 0,387898 0,051128 19,55883 -0,05529 0,831083
29 Gotham et al, 2018 PRC 55 -0,25 0,101032 9,897866 -0,873 0,372996
29 Gotham et al, 2018 PRC 56 -3,65 0,266782 3,748381 -4,66236 -2,63764
29 Gotham et al, 2018 PRC 57 1,5 0,128376 7,789641 0,797741 2,202259
29 Gotham et al, 2018 PRC 58 2,5 0,178376 5,606147 1,672203 3,327797
29 Gotham et al, 2018 PRC 59 3 0,212751 4,700339 2,095952 3,904048
29 Gotham et al, 2018 PRC 60 3,3 0,236376 4,230555 2,347078 4,252922
29 Gotham et al, 2018 PRC 61 3,85 0,285532 3,502236 2,80267 4,89733
29 Gotham et al, 2018 PRC 62 4 0,300251 3,330551 2,926015 5,073985
30 Laeng et al, 2018 PRC 63 0,3396 0,134223 7,450271 -0,37848 1,057675
31 Lynch et al, 2018 PLR 64 0,075445 0,183463 5,450699 -0,76407 0,914962
32 Nyström et al, 2018 PLR 65 0,896746 0,06531 15,3116 0,395852 1,39764
33 Aguillon-Hernandez et al, 2019 PRC 66 0,3408 0,067573 14,79886 -0,1687 0,850297
33 Aguillon-Hernandez et al, 2019 PRC 67 0,5698 0,035638 28,05988 0,19979 0,93981
35 Galazka et al, 2019 PRC 68 0,0962 0,034431 29,04352 -0,26749 0,45989
36 Li et al, 2019 PRC 69 0,2756 0,103605 9,65201 -0,35528 0,906481
37 Ring et al 2019 PRC 70 0,18 0,070655 14,15336 -0,34099 0,700986
38 Traynor et al, 2019 PRC 71 0,983469 0,169308 5,906408 0,176988 1,789951

of
38 Traynor et al, 2019 PRC 72 0,998545 0,169823 5,88849 0,190837 1,806252
38 Traynor et al, 2019 PRC 73 1,023311 0,170686 5,858706 0,213553 1,833069
38 Traynor et al, 2019 PRC 74 1,102073 0,173572 5,761287 0,285497 1,918648
38 Traynor et al, 2019 PRC 75 1,15964 0,175817 5,687726 0,337801 1,981479

ro
38 Traynor et al, 2019 PRC 76 1,152458 0,175531 5,697003 0,331289 1,973628
41 Granovetter et al, 2020 PRC 77 0,02 0,086961 11,49943 -0,55799 0,597987
41 Granovetter et al, 2020 PRC 78 0,04 0,086974 11,4977 -0,53803 0,61803
42 Hepach et al, 2020 PRC 79 -0,26812 0,073987 13,51591 -0,80125 0,26501
44
44
Reisinger et al, 2020
Reisinger et al, 2020
PRC
PRC
80
81
0,8289
0,497
-p 0,048343
0,046027
20,68547
21,72639
0,397953
0,076504
1,259847
0,917496
re
Table 3. (A) Baseline pupil size, (B) Latency, (C) Amplitude. Included effect sizes from each study, per
parameter, with corresponding variances, weights and 95% confidence intervals. Visual
representation in forest plots can be found in Figure 4. Study numbers relate to the same study
numbers as table 1 and 2.
lP
na
ur
Jo
 Baseline Latency Amplitude
Overall ES -0,0153 1,0262 -0,0123
se 0,2145 0,2571 0,1875
Variance - within = level 2 0,7501 0,3964 0,6383
Variance - between = level 3 0,3009 0,2779 0,5793
AIC full model 109,5451 55,0803 263,008
AIC reduced level 2 121,2615 73,3332 344,6245
AIC reduced level 3 108,3158 54,6664 295,5943
BIC full model 113,8471 57,9136 270,1913
BIC reduced level 2 124,1295 75,2221 349,4134
BIC reduced level 3 111,1838 56,5553 300,3832
LRT reduced level 2 13,7163 20,2529 83,6165
LRT reduced level 3 0,7707 1,5861 34,5863
Variance WITHIN YES YES YES

of
Variance BETWEEN NO NO YES

ro
Table 4. Evaluation of model fit. AIC= Akaike information criterion, BIC = Bayesian information
criterion, LRT= likelihood-ratio test

-p
re
lP
na
ur
Jo