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JournalsofofGerontology:
Gerontology:BIOLOGICAL
BiologicalSCIENCES
Sciences
Cite journal as: J Gerontol A Biol Sci Med Sci
Sci. 2012 November;67(11):11611169
doi:10.1093/gerona/gls080

The Author 2012. Published by Oxford University Press on behalf of The Gerontological Society of America.
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Advance Access published on March 28, 2012

Energy Balance Changes the Anabolic Effect of

Postexercise Feeding in Older Individuals
Brian D. Minor,1,* Daniel E. Heusinger,2,* Edward L. Melanson,3 Karyn L. Hamilton,1 and
Benjamin F. Miller1
1Department

of Health and Exercise Science and 2Department of Food Science and Human Nutrition, Colorado State University,
Fort Collins.
3Division of Endocrinology, Metabolism, and Diabetes, University of Colorado, Aurora.

*These authors contributed equally to this work.

We previously showed that consumption of protein immediately after exercise in older adults enhances nitrogen balance
when energy balance (EB) is maintained. Because daily EB routinely varies, it is important to know whether benefits
of postexercise protein consumption also occur with changing EB. Within an experiment, participants consumed an
isonitrogenousisocaloric diet with the timing of a protein (PRO + CHO) or carbohydrate (CHO) beverage immediately
after exercise versus earlier in the day. Within hypocaloric and hypercaloric cohorts, 3-day mean nitrogen balance was not
different when protein was consumed immediately after exercise, although there was a trend (p = .09) for higher nitrogen
balance in the positive EB. However, when data from our three studies were combined, the anabolic effect of postexercise
feeding was evident during positive EB but not negative EB. EB is therefore an important consideration in the postexercise
anabolic effect of protein feeding.
Key Words: NBALExerciseWasting.
Received November 1, 2011; Accepted February 13, 2012
Decision Editor: Rafael de Cabo, PhD

ITH age, there is a general wasting of proteins in

tissue (1). A multitude of factors contribute to the
wasting process; however, it is argued that the major contributors to the wasting process are a lack of physical activity
or disuse as well as inadequate calorie (2) and/or protein
intake (3,4). Regardless of the mechanism, wasting is the
result of a negative net protein balance, which occurs as
a result of decreased protein synthesis, increased protein
breakdown, or a combination of both.
Nitrogen balance (NBAL) is dependent on energy balance (EB) and protein intake. Energy restriction leads to a
decreased NBAL (5,6) because of increased amino acid
oxidation to meet energy needs. However, there is also evidence that increased protein intake is helpful in maintaining
NBAL. Specifically, consuming 0.92 gm of protein/kg body
weight (bw) has been shown to be more effective than 0.45 gm
of protein/kg bw at maintaining NBAL, lean body mass
(LBM), muscular strength, and immune function in elderly
women over the course of 9 weeks (7). However, even in
the presence of adequate protein intake, inadequate energy
intake results in lower NBAL than when EB is maintained
(8). Also, increasing energy intake will lead to an increased
NBAL due to protein sparing effects (9,10). Because protein synthesis is energetically costly (11) and is dependent
on ATP availability, elderly individuals who increase their

energy intake could reduce their dependence on amino acids

for energy purposes.
Exercise can also improve protein balance. Although
resistance training has been shown to increase muscle size
and strength in older individuals (12), many older individuals
are unable to perform resistance training due to prior injury,
limited access to equipment, and lack of knowledge regarding proper technique. Whole-body protein synthesis has
been shown to increase after a bout of aerobic exercise (3 h
at 75% VO2max), with whole-body protein breakdown not
differing from values at rest (13). A long-term aerobic exercise program has been shown to increase basal whole-body
protein turnover (14), whereas decreasing leucine oxidation
at rest (15). Additionally, it has been shown that prolonged
aerobic exercise can increase myofiber size in elderly
women (16), thus providing evidence of the benefits of
aerobic exercise at increasing LBM in elderly individuals.
The impact of aerobic exercise on NBAL is also dependent
on EB. When individuals maintain EB, aerobic exercise
increases NBAL over time (6,15). However, the increases
in NBAL observed with habitual aerobic exercise are
decreased when individuals are in a hypocaloric state (6).
Combining exercise with proper nutritional strategies has
been proposed to be more effective than either intervention
alone in stimulating muscle protein synthesis (MPS)
11611

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Address correspondence to Benjamin F. Miller, PhD, Department of Health and Exercise Science, Colorado State University,
200 Moby B Complex, Fort Collins, CO 80523-1582. Email: benjamin.f.miller@colostate.edu

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MINOR ET AL.

(17,18). Ingestion of amino acids with or without carbohydrate caused a shift toward increased protein balance
postexercise (17,19,20). However, by consuming amino acids with carbohydrates postexercise, net protein balance
was increased to levels greater than if the amino acids or
carbohydrate were ingested alone (19). In the elderly, protein combined with carbohydrate (40-g carbohydrate, 20-g
whey) led to increases in whole-body protein turnover
above those observed with an isoenergetic amount of carbohydrate (60-g carbohydrate; 21).
There is conflicting evidence as to whether timing of protein intake after exercise enhances protein synthesis and
thus LBM (22,23). We have addressed this question by
using NBAL and have shown that in older individuals in EB,
consumption of protein and carbohydrates (chocolate milk)
immediately following moderate aerobic exercise at 55%
VO2max led to greater NBAL than if consumed earlier in the
day (24). Findings from our previous study were interesting
given that differences in NBAL were noted with only the
timing of protein intake differing between trials. Because
many older individuals are habitually in negative EB (25),
and increasing energy intake leads to increases in NBAL
(9,10), we investigated the impact of the timing of protein
intake on NBAL in exercising older individuals in states of
hypocaloric and hypercaloric EB. We hypothesized that
timing protein intake after exercise in states of hypocaloric
and hypercaloric EB would enhance NBAL over values
observed when protein is consumed earlier in the day.

Methods

Study Overview
The study included two separate cohorts, hypocaloric
(n = 10) and hypercaloric (n = 6). In the hypocaloric cohort,
participants were in 15% negative EB for the duration of the
experimental period and participants in the hypercaloric
cohort were in 15% positive EB. Each experiment consisted
of four periods: preexperimental testing, a 7-day lead-in
diet, a 6-day inpatient/experimental period, and post-testing
(Figure 1A). Metabolic information collected during pretesting was used for planning the lead-in and experimental
diets and to determine the relative intensity of the daily
exercise during the experimental period. The 7-day lead-in
diet allowed participants to adapt to the level of dietary
protein that was provided during the experimental period.
All aspects of the study were completed in the same laboratory setting and repeated the design of our previously
published study (24). The study protocol was approved by
the Colorado State University Institutional Review Board
and the Colorado Multiple Institutional Review Board
for human research and adhered to the Declaration of
Helsinki.
Participants
Ten healthy sedentary men (n = 2) and women (n = 8)
participated in the hypocaloric cohort, and six healthy sedentary men (n = 2) and women (n = 4) participated in the

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Figure 1. Study timeline (A) and daily timing of meals, exercise, and consumption of beverages during PRO + CHO and CHO trials (B). The timeline in panel B
occurs within the period labeled Experimental in panel A. The order of the PRO and CHO trials was randomized.

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ANABOLIC EFFECT
EFFECT

Table 1. Participant Characteristics of Both Energy Balance Cohorts

Hypocaloric Cohort

Age (years)
Height (cm)
Weight (kg)
Body mass index (kg/m2)
Body fat (%)
VO2max (mL/kg/min)

Hypercaloric Cohort

Men (n = 2)

Women (n = 8)

All Participants (n = 10)

Men (n = 2)

Women (n = 4)

All Participants (n = 6)

67 1
183 1
93 1
27.4 0.3
27.9 1.0
32.2 0.5

63 2
165 3*
61 2*
22.3 0.6*
34.5 1.7
28.5 1.7

64 2
169 3
67 4
23.3 0.8
33.2 1.7
29.2 1.4

63 6
178 5
85 3
26.9 2.5
24.7 1.3
29.4 2.1

66 2
160 2*
60 3*
23.5 1.8
37.9 4.2
21.1 1.2*

65 2
166 4
68 6
24.6 1.5
33.5 3.9
23.9 2.0*

Note: *Significant difference between hypocaloric and hypercaloric cohorts.

Pretesting
Over four separate days, participants completed a series
of pretesting at the Human Performance Clinical/Research
Laboratory at Colorado State University. During the initial
visit, participants completed a Balke protocol graded exercise test on a treadmill under the supervision of a cardiologist. Participants were excluded if the graded exercise test
indicated a cardiac ischemic or hypertensive response to the
exercise. Participants returned to the Human Performance
Clinical/Research Laboratory after an overnight fast, and
resting metabolic rate (RMR; Parvomedics TrueOne 2400,
Sandy, UT) was measured to determine 24-hour resting
caloric expenditure. During the test, participants rested in
the supine position for 45 min and were instructed to remain
still and to refrain from sleeping. The first 15 min of the test
was used to achieve the appropriate flow rate and to allow
participants to become familiarized with the experimental
conditions. The data from the final 30 min of the test were
used to predict RMR. Body composition was then determined using dual-energy X-ray absorptiometry (QDR
4500W, Hologic, Inc., Bedford, MA). On the third day of
testing, participants completed an incremental exercise test
on a cycle ergometer (Monark Excalibur, Groningen, The
Netherlands) with indirect calorimetry (Parvomedics TrueOne 2400) to determine maximal oxygen consumption
(VO2max). Participants pedaled at 50 watts for the first minute,
and watts increased by 20 for women and 30 for men every
2 min thereafter (24). The test was stopped when participants reached volitional exhaustion. After VO2max was
determined, 55% of each participants VO2max was calculated using the American College of Sports Medicine leg
cycle ergometry equation (26).

On a subsequent day, participants returned to the Human

Performance Clinical/Research Laboratory for a submaximal
steady-state cycling test in which participants cycled at their
estimated workload (55% VO2max) for 45 min. Energy
expenditure (EE) was determined by averaging the EE from
the final 30 min of cycling using indirect calorimetry. The
calculated EE from the 30 min of cycling was used to estimate
the participants exercise EE in kcal/hour for the 1 hour of
cycling exercise during the inpatient period.
During pretesting, participants completed an online food
preference and food allergy questionnaire from the Clinical
and Translational Research Center (CTRC) to ensure that
all study diets contained acceptable foods. Participants also
recorded all foods and beverages that were consumed over
3 consecutive days (2 weekdays and 1 weekend) in a 3-day
diet log. All foods from the 3-day diet records were entered
into Nutrition Pro software (Axxya Systems, Stafford, TX)
and analyzed to determine habitual (free-living) energy and
macronutrient intake.
Lead-in Diet
Following completion of the pretesting, participants completed a controlled 7-day lead-in diet immediately followed
by a 6-day inpatient stay at the University of Colorado
Denver CTRC. The lead-in diet allowed participants to adapt
to a new level of protein intake, so that NBAL measurements made during the inpatient stay would not reflect an
acute adaption to a new level of protein intake. During the
lead-in diet period, participants arrived every morning at the
Colorado State University Nutrition Center for breakfast.
Participants ate breakfast at the nutrition center under the
supervision of study staff. Food for the remainder of the day
was prepared and given to the participants in a cooler.
Participants were instructed to eat only the food given to them
by the study staff and to eat all of the food given to them. If
the participants were unable to eat any of the food provided
to them, they brought the food back with them the following
day so that it could be weighed. Participants were instructed
to continue their typical daily activities throughout the duration of the 7-day lead-in period. The lead-in and inpatient
diets followed U.S. Department of Agriculture nutritional
guidelines (27) and were constructed using Pronutra software
(Viocare, Inc., Princeton, NJ). Participants remained in EB
for the duration of the lead-in diet for both the negative and

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hypercaloric balance cohort as depicted in Table 1. All participants were nonsmokers between the ages of 55 and
75 years and did not take any medications. Participants
were required to be lactose tolerant because milk was used
as an experimental beverage. Additional exclusion criteria
included obesity (body mass index > 30), recent orthopedic
injury that would impede the ability to exercise, any condition that affected food digestion or absorption, a thyroid
condition (thyroid stimulating hormone <0.05 uU/mL or
thyroid stimulating hormone > 5.0 uU/mL), or any current
illness or infection.

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MINOR ET AL.

Experimental/Inpatient Period
The experimental period involved a 6-day inpatient stay
at the University of Colorado Denver CTRC (Figure 1B).
Each participant completed 2 consecutive, 3-day trials in a
randomized crossover design. The diets for each 3-day trial
were reproduced and identical in calorie intake, macronutrients, and foods consumed. The diet plans for Day 1, 2, and
3 were repeated on Day 6, 4, and 5, respectively. The only
difference between trials was the timing of intake of the
protein beverage. For the negative EB cohort, participants
were in a 15% caloric deficit, and similar to the lead-in diet,
the percentage of total kilocalories for each macronutrient
in the inpatient diet was 55% carbohydrate, 30% fat, and
15% protein. Participants in the positive EB cohort were in
positive 15% EB and, similar to the lead-in diet, the inpatient
diet consisted of 1.2-gm protein/kg bw, 30% of calories
from fat, and the balance as carbohydrate.
Participants were admitted to the CTRC the evening
before the 6-day inpatient period and provided dinner as the
final meal of their lead-in diet. The study began the following morning with the start of the study diet and 24-hour
urine collection. Daily weight measurements were recorded
each morning on the same scale after voiding. Day 1 and
Day 6 were spent in a whole-room calorimeter, whereas
days 25 were spent in a patient room on the CTRC. During
days 25, participants were permitted to leave the CTRC
unit twice a day for 30 min. Participants were allowed to eat
only the food that was provided to them by study staff and
to consume water ad libitum. Every day at 4:30 pm, participants completed 1 hour of cycling exercise (Lode, Groningen,
The Netherlands) at 55% of their VO2max. The exercise was
intended to simulate a brisk walk, which was well tolerated
by all participants. Participants were not permitted to engage
in any additional volitional exercise during the day.
Immediately following the daily exercise bout, a postexercise beverage was consumed. The only difference between
the two trials was the timing of intake of the two experimental beverages. In one trial (PRO + CHO), a 248-kcal chocolate milk drink that contained 15.3-gm protein, 43.6-gm
carbohydrate, and 1.3-gm fat (330-gm skim milk, 4-gm
whey protein, and 42-gm chocolate syrup) was consumed

immediately postexercise. In the second trial (CHO), a 247kcal carbohydrate beverage that contained 0.0-gm protein,
63.51-gm carbohydrate, and 0.06-gm fat was consumed
immediately postexercise. During the PRO + CHO trial,
the CHO beverage was consumed as a snack at 10 am and
during the CHO trial, the PRO + CHO beverage was consumed
as a snack at 10 am.
In order to plan the diets for the inpatient period, total
daily EE was estimated. Each participants RMR was multiplied by a separate activity factor for calorimeter and noncalorimeter days. The activity factor was lower for calorimeter
days because ambulatory activity levels are lower when confined to the calorimeter room (E. L. Melanson, PhD, unpublished data, 2010). Exercise EE was estimated from the
steady-state VO2 data (measured during pretesting), and an
additional 20% of exercise calories were added in order to
account for excessive postexercise oxygen consumption.
Participants entered the calorimeter room at 7:45 am on
Day 1 and Day 6 and exited at 7:15 am on the following
morning. The calorimeter room was 12 12 and contained
a bed, sink, toilet, bicycle ergometer, computer, and television.
To prevent air from escaping, all meals were passed through
an air lock that could not be simultaneously opened from the
inside and outside. Daily EE and substrate oxidation were
calculated by the difference in gas content that was entering
and exiting the room. EE and respiratory quotient were
assessed in 1-minute intervals from oxygen consumption
and carbon dioxide production. Gas concentrations were
determined from the flow rate and the differences in CO2
and O2 concentrations between entering and exiting air by
using a fuel-cellbased dual channel O2 analyzer (FC-2
Oxzilla, Sable Systems, International, Las Vegas, NV) and
two infrared CO2 analyzers (CA-10 CO2 analyzers, Sable
Systems, International), as previously described (29). The
accuracy and precision of the system are tested monthly
using propane combustion tests. The average O2 and CO2
recoveries during the course of this study were 98.0%. Total
EE and substrate oxidation were calculated using oxygen
consumption and respiratory quotient (30). Urine N was
used to calculate 24-hour protein oxidation, with 1 gm of
urine N reflecting 6.25 gm of oxidized protein.
During the inpatient stay, participants wore an accelerometer (Actigraph GT1M, Pensacola, FL), which was
removed during exercise, sleep, and showering. EE from
activity was estimated from the accelerometer. If activity
counts were less than or equal to 1,952/min, the Work
Energy Theorem was used to estimate EE:
Kcal/minute = (0.0000191) (counts/minute)
(body mass [kg]).

The Freedson equation was used to estimate EE for activity

counts greater than 1,952/min:
Kcal/minute = (0.00094) (counts/min)
+ (0.1346) (body mass [kg]) (7.37418).

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positive EB cohorts. During the negative EB cohort, the

lead-in diet (and all inpatient diets) macronutrient breakdown was 15% protein, 30% fat, and 55% carbohydrate
expressed as a percentage of total calories. During the positive EB cohort, the lead-in diet (and all inpatient diets)
consisted of 1.2-g protein/kg bw, 30% of calories from
dietary fat, and the balance as carbohydrate. We used an
absolute quantity of protein in the hypercaloric cohort to
match the hypocaloric cohort protein intake. For the lead-in
diets, total energy intake was calculated using each participants RMR, multiplied by an activity factor, which
approximated the activity levels of free-living sedentary elderly
individuals (28).

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Table 2. Energy Intake, EE, and EB in Hypo- and Hypercaloric Cohorts

Macronutrient Intake (gm/kg bw)
Protein

Hypocaloric
Hypercaloric

CHO

Fat

Free Living

Lead in

Inpatient

Free Living

Lead in

Inpatient

Free living

Lead in

Inpatient

1.27 0.11
1.13 0.09

1.16 0.04
1.23 0.07

1.07 0.04
1.20 0.00

3.87 0.37
3.24 0.23

4.23 0.15
4.53 0.27*

3.91 0.14
5.87 0.36*#

1.18 0.11
1.13 0.14

1.03 0.04
1.10 0.06

0.95 0.03
1.35 0.07

EB
Energy Intake (kcal)
Free Living

Lead in

2,016 136
1,914 180

2,003 122
2,160 161

Inpatient
1,860 126
2,665 224*

Nonexercise PAL

Mean Energy Balance (kcal)

CHO

PRO + CHO

CHO

PRO + CHO

CHO

PRO + CHO

1.60 0.02
1.59 0.04

1.61 0.02
1.57 0.04

1.36 0.02
1.37 0.02

1.37 0.03
1.35 0.03

285 35
360 51

291 40
354 88

Notes: bw = body weight; EB = energy balance; EE = energy expenditure; and PAL = physical activity level.
*Significant difference from free-living conditions within the same cohort.
#Significant difference from lead-in conditions within the same cohort.

Total daily energy expenditure (TDEE) on calorimeter

days was directly measured, and noncalorimeter days were
estimated:
TDEE = RMR + DIT + activity thermogenesis.

Because participants were sedentary, the EE predicted by

the accelerometer was used for activity thermogenesis. The
measured value for RMR was used, and DIT was estimated
to be 10% of TDEE (31). Physical activity level was calculated by dividing TDEE by RMR. EB was determined using
calorimeter room EE and energy intake, which was calculated from the diet plans created by the dietitian.
Nitrogen Balance
Twenty-four-hour urine samples were collected continuously beginning with the first void on Day 1 in order to
determine urinary nitrogen. Urine was collected in acid, and
total volume was measured. Two 10-mL aliquots per 24hour collection were frozen and stored for later analysis.
Nitrogen was analyzed using an Antek 7000 Elemental
Nitrogen Analyzer (PAC, Houston, TX). NBAL was calculated as

Laboratory for a final dual-energy X-ray absorptiometry

scan and weight measurement.
Statistical Analysis
To provide further insight into the effect of EB on the
anabolic effect of postexercise feeding, we combined data
from these two corhorts and our previously published study
(24). Although mean data in each EB group (negative, balance, and positive) were in our target range, there was variability within each group leading to real variability. We feel
this further analysis was justified because all studies were
conducted under identical conditions. The additional insight
gained can only be captured in the combined data; however,
the data are presented separately so the reader can make that
assessment his/herself.
NBAL data, EB data, and participant characteristics were
analyzed using students paired t-tests. A one-way analysis
of variance with a Student NewmanKeuls post hoc analysis
was used to examine nutrient intake during the different
periods of testing. Differences were considered significant
at p < .05. All data are presented as the mean SEM unless
indicated otherwise.

NBAL (gm) = nitrogen intake

(urinary N + fecal N losses + miscellaneous N losses).

Nitrogen intake was calculated as (protein intake [gm]/6.25)

because 6.25 gm of protein contains on average 1 gm of
nitrogen. Nitrogen output is defined as the N excreted in
urine and feces plus miscellaneous N losses. Daily miscellaneous N losses were estimated at 5 mg/kg bw, and fecal N
losses were estimated at 2 gm (32). Three-day mean NBAL
from each trial (PRO + CHO and CHO) was compared with
test the hypothesis that timing protein intake after aerobic
exercise improves NBAL in older adults.
Post-testing
Approximately 1 week after the inpatient period, participants
returned to the Human Performance Clinical/Research

Results

Macronutrient Intake and EB

There were no differences in macronutrient intake except
for the hypercaloric diet in which carbohydrate intake was
increased during the inpatient period (Table 2). During the
inpatient period in the hypocaloric cohort (Table 2), participants were in negative EB, with a mean daily negative EB
of 285 35 kcal during the CHO trial and 291 40 kcal
during the PRO + CHO trial (p = .90). During the inpatient
period in the hypercaloric cohort, participants had an
increased energy intake from free-living conditions that
resulted in a positive EB, with a mean daily positive EB of
360 51 kcal for the CHO trial and 354 88 kcal for PRO +
CHO trial (p = .98). Mean bw did not change during the

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Hypocaloric
Hypercaloric

PAL

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MINOR ET AL.

inpatient stay in either hypocaloric or hypercaloric cohorts

(Figure 2).
Nitrogen Balance
In the hypocaloric cohort, 3-day mean NBAL was not
different between CHO and PRO + CHO trials (mean
SEM, 95% CI for CHO: 0.07 0.52, 1.25 to 1.11; for
PRO + CHO: 0.09 0.53, 1.09 to 1.29; p = .28; Figure 3A).
In the hypercaloric cohort, 3-day mean NBAL was not
different between the CHO and the PRO + CHO trials (mean
SEM, 95% CI for CHO: 0.97 0.52, 0.36 to 2.30; for PRO +
CHO: 1.66 0.43, 0.55 to 2.76), although there was a trend
toward significance (p = .09; Figure 3A). In both cohorts,
there was no difference in NBAL between CHO and PRO +
CHO in days 13 and days 46 (p = .25 for negative EB and
p = .35 for positive EB; Figure 3B).
Combined Data for Negative, Even, and Positive EB
Cohorts
When values from all trials together without stratification
for EB were compared, mean NBAL was greater for PRO +
CHO compared with CHO (CHO: 0.42 0.29 gm N, 0.19
to 1.03 gm N; PRO + CHO: 0.85 0.29 gm N, 0.25 to 1.45 gm
N; p < .01; Figure 4A). We then restratified all the combined
data so that 6-day mean EB percentage above 0.0 was
placed in the positive group and below 0.0 were placed in
the negative group. The resultant adjusted model had a
greater percent EB in the positive EB group compared with
the negative EB group (negative: 13.29 2.41%, 18.85 to
7.74%; positive: 12.85 1.96%, 8.60 to 17.09%; p <
.0001; Figure 4B). In the restratified analysis, NBAL was
greater in the positive EB groups compared with the negative
EB in both the CHO trial (negative: 0.43 0.42 gm N, 1.39
to 0.53 gm N; positive: 0.96 0.34 gm N, 0.25 to 1.69 gm
N; p = .009) and the PRO + CHO trial (negative: 0.15
0.47 gm N, 1.24 to 0.94 gm N; positive: 1.49 0.26 gm N,

Figure 3. Nitrogen balance in negative and positive energy balance (EB)

groups. There were no differences between CHO and PRO + CHO groups in
either negative EB (p = .28) or positive EB although there was a trend (p = .09; A).
There was no order effect of treatment on NBAL (p = .25 for negative EB and
p = .35 for positive EB; B).

0.94 to 2.05 gm N; p = .002; Figure 4C). There was no difference in mean NBAL between the CHO and PRO + CHO
trial in the negative EB group (CHO: 0.43 0.42 gm N,
1.39 to 0.53 gm N; PRO + CHO: 0.15 0.47 gm N,
1.24 to 0.94 gm N; p = .18; Figure 4D). However, mean
NBAL was greater (p = .016) in the PRO + CHO compared
with CHO in the positive EB group (CHO: 0.96 0.34 gm
N, 0.25 to 1.69 gm N; PRO + CHO: 1.49 0.26 gm N, 0.94
to 2.05 gm N; p = .016; Figure 4D).
Discussion
The current studies investigated the effects of the timing
of protein intake in relation to a bout of aerobic exercise on
NBAL in older individuals with varying energy intakes. Older
individuals completed two 3-day isocaloricisonitrogenous
trials with only the timing of protein consumption differing
between the two conditions. Contrary to our initial hypotheses, NBAL did not differ in older individuals on either
hypocaloric or hypercaloric diets when protein was consumed immediately after moderate aerobic exercise rather
than earlier in the day. However, further stratification of
positive versus negative EB to account for EB variations
within a trial revealed a strong influence of EB on the anabolic effect of postexercise feeding. To our knowledge, this
study is unique given its aim to investigate the combined

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Figure 2. Mean daily body weight during inpatient period for both negative
energy balance (EB; n = 10) and positive EB (n = 6) groups (mean SEM).

 EB
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CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT

11677

effects of varying EB and the timing of protein intake on

NBAL in older individuals.
Energy Balance and Nitrogen Balance
NBAL is dependent on EB such that NBAL is better
maintained when caloric intake is adequate and negative
NBAL results when energy intake is reduced (6). During
caloric restriction, protein breakdown increases the availability of amino acids that are oxidized as energy and thus
limits the availability of amino acids for protein synthesis.
When caloric intake is inadequate, aerobic exercise can
increase NBAL (6). Consistent with this, within the hypocaloric cohort of the current study older individuals were able
to maintain NBAL for both the CHO and PRO + CHO trials
despite being in negative EB. Because loss of LBM occurs
when individuals are in negative NBAL (5), maintaining
NBAL while in negative EB could reduce the loss of LBM
and thus attenuate the progression of wasting.
Within the negative EB cohort, there was no differences
in NBAL between the CHO and PRO + CHO trials, which
differs from previous research by Roy and colleagues (33).
In that study, participants underwent two 7-day trials in
which they increased their training volume and consumed
either a mixed-meal beverage or a noncaloric placebo beverage after exercise. Participants were in an exerciseinduced negative balance, as calories expended during
exercise were not replaced. Similar to our study, diet was

replicated so that the only difference between the trials was

the timing of consumption of the beverage. Timing nutrient
intake immediately after exercise, rather than earlier in the
day, resulted in greater NBAL with a trend toward significance (p = .06; 33). However, the participants in the Roy
study were young female athletes whereas the present study
was in older individuals, exercise was performed every
other day rather than every day, and NBAL only determined
over the last 24 h as opposed to every day.
Within the restratified negative EB group, there were no
differences in NBAL between the PRO + CHO and CHO
trials. Although this was consistent with the results from the
negative EB cohort, these results differ from previous research by Roy and colleagues (33). In the positive EB
group, there was increased protein retention in the PRO +
CHO compared with the CHO trials. Thus, our data indicate
that timing protein intake after exercise has a greater impact
on improving NBAL when older individuals are in positive
EB rather than negative EB.
Within the restratified EB groups, mean NBAL was
greater in the positive EB groups compared with negative
EB for both CHO and CHO + PRO treatments. Previously
Howarth and colleagues (34) demonstrated that consuming
protein with carbohydrate after aerobic exercise increases
NBAL more than an isocaloric amount of only carbohydrate. The differences between EB groups demonstrate that
EB alone, a factor often not controlled for, can be increase

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Figure 4. Combined data from all three studies to determine the effect of energy balance (EB) on nitrogen balance. When combining all data without regard
to EB, NBAL was greater in PRO + CHO compared with CHO (p < .01; A). Restratified negative EB was lower compared with positive EB (p < .0001; B).
When restratified by treatment, positive EB had greater NBAL in both CHO (p = .009) and PRO + CHO groups (p = .002; C). When restratified by EB, there was no
increase with PRO + CHO in the negative EB group (p = .18), but there was an increase with PRO + CHO in the positive EB group (p = .016; D).

81168

MINOR ET AL.

Experimental Diet and Exercise

The study was designed to have practical implications.
All study diets followed U.S. Department of Agriculture
dietary recommendations for the percentage of total kilocalories for each macronutrient (4565% carbohydrate, 2035%
fat, and 1035% protein; 27). The daily exercise completed
by participants during the inpatient stay was 1 hour of moderate intensity (55% VO2max) cycling. The exercise intensity
and duration were well-tolerated by all participants, and the
inpatient physical activity level was consistent with older
populations (28). Additionally, exercise at 55% of VO2max
simulates a brisk walking pace because older individuals
most commonly choose walking as exercise (37).
Chocolate milk was chosen for this study as the protein
source because milk contains a high quantity of leucine and
consists of both whey and casein proteins (38). Older individuals have a blunted MPS response to anabolic signals
compared with younger individuals (39,40). However,
increasing the proportion of leucine allows for optimal
stimulation of MPS in older individuals (41). Whey protein
results in an acute increase in protein synthesis (42),
whereas casein protein results in a minor increase in protein

synthesis and a significant decrease in protein breakdown

(42). Previously, it was shown that consumption of carbohydrate combined with protein after aerobic exercise increases
whole-body net protein balance and muscle FSR more than
an isocaloric amount of carbohydrate alone (34). The current
study indicates that this is true when in positive EB conditions but not necessarily negative EB conditions.
Limitations
Inadequate accounting of protein intake or miscellaneous
nitrogen losses can contribute to variability in the NBAL
method (43). However, the same foods were consumed
during both trials and were closely monitored so any measurement error would occur during both conditions to the
same extent. In addition, miscellaneous protein losses were
held constant between trials, and we have no reason to
believe that would vary. Therefore, any differences in NBAL
estimations would be consistent between the two trials.
Despite limitations, the NBAL technique can be a useful
technique for studying whole-body protein retention in
aging populations. The NBAL technique with its noninvasive
nature makes it a relatively easy and appropriate method to
determine long-term changes in whole-body protein retention, an important outcome when considering wasting, in
response to exercise and nutrition interventions in older
individuals.
Conclusions and Recommendations
The current study investigated whether having a proteincontaining beverage immediately after moderate aerobic
exercise rather than earlier in the day can improve NBAL in
older individuals with varying EB. The combined results
from the current study and our previous study (24) indicate
that EB is an important determinant of the anabolic effect of
protein feeding and therefore must be considered when
using the NBAL method. We demonstrated that nitrogen
retention is greater during positive EB than negative EB and
that the beneficial effects of a protein-containing beverage
on nitrogen retention are present with positive EB but are
negligible during negative EB. Future studies should consider the effect of EB on the timing of protein intake after
exercise. Further, it may be important to independently assess skeletal muscle and whole-body outcomes because
they may not be the same.
Funding
This work was funded by the Colorado Agriculture Experimental
Station (grant #COL00604), a career development support from NIA
K01AG031829-01 (B.F.M.), and the UCD Clinical and Translational
Science Award (1UL1 RR025780).
Acknowledgments
The study participants are thanked for their exceptional dedication.
Further, we thank Archana Mande for designing the study diets and the
University of Colorado Denver staff for their help.

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NBAL (9,10). We therefore recommend consideration of

EB when performing short- and long-term studies of protein
balance.
Because the NBAL method does not elucidate rates of
protein synthesis or breakdown, we were unable to determine how changes in net NBAL occurred. Also, it is not
known where protein accrual is taking place. It could be that
MPS increased in response to the exercise stimulus (35).
However, because skeletal muscle contributes approximately
30% of the whole-body protein turnover rate (36), changes
in whole-body protein, outside of skeletal muscle, are also
expected to contribute to changes in protein balance. Interestingly, in a recent study in our laboratory, we found in a
similar group of older individuals that postexercise feeding
of PRO + CHO versus CHO did not result in increased
cumulative MPS over a 6-week aerobic exercise-training
program (22). There could therefore be differences in wholebody protein retention versus skeletal MPS. NBAL accounts
for whole-body protein retention whereas our previous study
examined MPS. This relative long-term difference (compared with an acute feeding) in whole-body versus MPS in
response to postexercise feeding has still not been adequately
addressed. Alternatively, in our previous study (22), EB was
not controlled for the 6-week period as the participants were
free living and were only encouraged to maintain EB. The
current study only demonstrated a positive effect of PRO + CHO
compared with CHO in positive EB; therefore, day-to-day
variations in EB may change whether protein is accrued or
not. These differences illustrate the importance of considering
EB when examining skeletal muscle (sarcopenia) or wholebody (wasting) outcomes.

 EB
EB CHANGES
CHANGES ANABOLIC
ANABOLIC EFFECT
EFFECT

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