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Research Paper

Altering the rest interval during high-intensity interval training does not affect muscle or performance adaptations
Johann Edge1,2 , Nir Eynon3,4 , Michael J. McKenna3 , Craig A. Goodman4 , Roger C. Harris5 and David J. Bishop3,4
1 2

Muscle Metabolism Laboratory, Department of Sport and Exercise Science, University of Auckland, Auckland, New Zealand School of Human Movement and Exercise Science, The University of Western Australia, Crawley, Western Australia, Australia 3 Institute of Sport, Exercise and Active Living (ISEAL) and 4 School of Sport and Exercise Science, Victoria University, Melbourne, Australia 5 School of Sport and Exercise Science, University of Chichester, Chichester, UK

Experimental Physiology

New Findings r What is the central question of this study? Are exercise-induced changes in metabolites and ions a crucial factor in the adaptation of contracting muscle? This was assessed by manipulating the rest period between high-intensity intervals. r What is the main nding and its importance? Our results suggest that the perturbation of muscle metabolites (specically phophocreatine, lactate and H+ ) during high-intensity interval training is not a crucial factor regulating related adaptations of the contracting muscle, when training intensity and volume are matched. This has implications for understanding the mechanisms that regulate muscle adaptations. It has been hypothesized that exercise-induced changes in metabolites and ions are crucial in the adaptation of contracting muscle. We tested this hypothesis by comparing adaptations to two different interval-training protocols (differing only in the rest duration between intervals), which provoked different perturbations in muscle metabolites and acidbase status. Prior to and immediately after training, 12 women performed the following tests: (1) a graded exercise O2 peak ); (2) a high-intensity exercise bout (followed 60 s test to determine peak oxygen uptake (V later by a repeated-sprint-ability test; and (3) a repeat of the high-intensity exercise bout alone with muscle biopsies pre-exercise, immediately postexercise and after 60 s of recovery. Subjects performed 5 weeks (3 days per week) of training, with either a short (1 min; HIT-1) or a long rest period (3 min; HIT-3) between intervals; training intensity and volume were matched. Muscle [H+ ] (155 15 versus 125 8 nmol l1 ; P < 0.05) and muscle lactate content (84.2 7.9 versus 46.9 3.1 mmol (g wet weight)1 ) were both higher after HIT-1, while muscle phosphocreatine (PCr) content (52.8 8.3 versus 63.4 9.8 mmol (g wet weight)1 ) was lower. There were no O2 peak , repeatedsignicant differences between the two groups regarding the increases in V + + sprint performance or muscle Na ,K -ATPase content. Following training, both groups had a signicant decrease in postexercise muscle [H+ ] and lactate content, but not postexercise ATP or PCr. Postexercise PCr resynthesis increased following both training methods. In conclusion, intense interval training results in marked improvements in muscle Na+ ,K+ -ATPase content,

Sadly, J. Edge is deceased, 25 March 2010.

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DOI: 10.1113/expphysiol.2012.067603

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O2 peak . However, manipulation of the rest period during intense interval PCr resynthesis and V training did not affect these changes.
(Received 3 June 2012; accepted after revision 21 August 2012; rst published online 23 August 2012) Corresponding author D. J. Bishop: Institute of Sport, Exercise and Active Living (ISEAL), Room 140, Building P, Footscray Park campus, Victoria University, Melbourne, Victoria, Australia. Email: david.bishop@vu.edu.au

Intense exercise induces marked elevations in ATP utilization and provokes considerable metabolic and ionic perturbations, resulting in elevations in the muscle content of lactate (MLa) and H+ ions, as well as reductions in ATP and phosphocreatine (PCr; McKenna et al. 2008). Altered concentrations of these metabolites and ions have been implicated in the multifactorial process of muscle fatigue, owing to their effects on muscle enzyme activity, sarcoplasmic reticulum Ca2+ release and cellular membrane excitability (Allen et al. 2008; McKenna et al. 2008). The restoration of perturbed metabolite and ion concentrations towards resting levels following intense muscle contractions is likely to be important for the recovery of subsequent exercise performance. In support of this, the resynthesis of PCr has been related to the recovery of force output during repeated sprints (Bogdanis et al. 1996), and the PCr resynthesis rate is elevated in trained compared with untrained muscle (Bishop et al. 2008). Therefore, improving the capacity to minimize exercise-induced disturbances in these metabolites and ions during high-intensity exercise, as well as maximizing their rate of recovery, may be important to enhance performance. Training induces many adaptations within skeletal muscle that minimize these metabolic and ionic disturbances during exercise and enhance their postexercise recovery towards resting levels (Harmer et al. 2000; Juel et al. 2004; Burgomaster, 2006; Edge et al. 2006b). It has previously been hypothesized that exerciseinduced changes in metabolites and ions are a crucial factor in the adaptation of contracting muscle, and that the above-mentioned adaptations might be greater with training that provokes greater changes in metabolites and ions (Weston et al. 1997; Mohr et al. 2007). In support of this, it has been reported that running training that induced greater elevations in venous [K+ ] and [H+ ] was associated with greater increases in abundance of Na+ ,K+ ATPase isoforms and the Na+ H+ exchanger (Mohr et al. 2007); however, in that study neither training intensity nor volume was matched. It is therefore impossible to attribute these ndings solely to differences in the intramuscular accumulation of MLa, H+ and K+ during exercise. An alternative, and novel, experimental approach to augment the metabolic and acidbase disturbances during exercise, while matching training volume and intensity, is to restrict the rest period between exercise intervals. Decreasing the rest period between intervals during training results in larger changes in muscle pH, MLa

and PCr levels (Yoshida & Waitari, 1993). Thus, if the intramuscular metabolic or acidbase disturbances during exercise are indeed an important training stimulus, one would hypothesize greater improvements in the regulation of metabolites and ions following interval training that employs short compared with long rest intervals. In the present study, therefore, we used two different highintensity exercise training protocols designed to provoke different perturbations in muscle metabolites and acid base status, to examine the effects on physiological and performance adaptations to training. We hypothesized that interval training with a shortened recovery period would exacerbate metabolic and acidbase disturbances and thereby augment subsequent training adaptations. Methods
Ethical approval

Informed, written consent was obtained from each participant. Approval for the study procedures was granted by the University of Western Australia Research Ethics Committee. The study was conducted according to the Declaration of Helsinki.
Subjects

Twelve women (19 1 years old; mean SD) gave written informed consent and participated in this study. Each subject was involved in an intermittent sport at club level (hockey, tennis, netball, basketball or football), in which they continued to participate throughout the study (training 23 days a week and match play 1 day each week).
Experimental overview

Pre- and post-training, participants performed the following three tests, on separate days, in the same order, as follows: (1) a graded exercise test (GXT) to determine O2 peak ); (2) a 45 s continuous highpeak oxygen uptake (V intensity exercise bout at a work rate corresponding to O2 peak (HIE) followed 60 s later 200% of the pretraining V by a repeated-sprint-ability (RSA) test (HIE + RSA); and (3) a repeat of the HIE (without the subsequent RSA test), during which muscle biopsies were obtained pre-exercise, immediately postexercise and after 60 s of recovery (Fig. 1).
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Pre- and post-training tests were conducted at the same time of day to avoid possible time-of-day effects (Racinais et al. 2005; Racinais et al. 2010). Subjects were asked to maintain their normal diet and training throughout the study. They were also required to consume no food or beverages (other than water) for 2 h prior to testing and were asked not to consume alcohol and caffeine or to perform vigorous exercise in the 24 h prior to each test. Food diaries were given to each subject to record food and uid consumption for 2 days prior to each test, and subjects were asked to replicate this during post-training testing.
Graded exercise test

The GXT was performed on an air-braked track-cycle ergometer (Evolution Pty Ltd, Adelaide, South Australia, Australia) and consisted of 4 min stages, with a 1 min break between stages (Bentley et al. 2007). Both the O2 peak were determined from lactate threshold (LT) and V the GXT. During the GXT, expired air was analysed continuously for O2 and CO2 concentrations using Ametek gas analysers (SOV S-3A11 and COV CD-3A; Applied Electrochemistry, Pittsburgh, PA, USA), and ventilation was recorded every 15 s using a turbine ventilometer (225A; Morgan, Rainham, UK). The gas analysers were calibrated immediately before and veried after each test using three certied gravimetric gas mixtures (BOC Gases, Chatswood, NSW, Australia); the ventilometer was calibrated pre-exercise and veried postexercise using a 1 litre syringe in accordance with the manufacturers instructions. The LT was calculated using the modied Dmax method (LTDmax ; Bishop et al. 2000).
High-intensity exercise bout (45 s constant cycling) and repeated-sprint-ability test

and after exercise at a designated workload, as well as the capability to recover from a bout of high-intensity exercise and subsequently perform repeated sprints. The HIE consisted of 45 s of continuous cycling on an airbraked, front-access cycle ergometer (Model Ex-10; Repco, Sydney, Australia). Toe-clips and heel-straps were used to secure the feet to the pedals, and the test was performed in the seated position. The power output (in watts) for both the pre- and post-training HIE was 200% of the O2 peak . At the pretraining power output that elicited V completion of the HIE, subjects remained on the cycle ergometer, and 60 s later they performed an RSA test comprised of ve maximal sprints of 6 s duration, repeated every 30 s (Mendez-Villanueva et al. 2008). During the 24 s recovery between sprints, subjects rested on the ergometer. Five seconds before starting each sprint, subjects were asked to assume a standing position and to await the start signal. Strong verbal encouragement was provided to each subject during all sprints. Performance measures during the RSA test included peak power and work output for each sprint, as well as total work for all ve sprints. The trials with muscle biopsies were performed on a separate day in order not to interfere with the short recovery period (60 s) or repeated-sprint performance. On the day of muscle biopsy, the exact procedure was followed as for the HIE test, except that muscle biopsies were taken at rest (lying in the supine position) and then again immediately and 60 s following the HIE (i.e. the RSA test was not performed on this day). The subject remained seated on the ergometer for both of the postexercise samples.
Training intervention

This combined test (HIE + RSA) was designed to assess the effects of training on changes in metabolites during

Following baseline testing, subjects were matched on their LTDmax and then randomly allocated to a high-intensity interval-training programme employing either 1 (HIT-1) or 3 min (HIT-3) passive rest periods between intervals. Within 47 days of baseline testing, all subjects started

Figure 1. Experimental overview O peak ) and the lactate Denitions: GXT, graded exercise test to determine peak oxygen uptake (V 2 O peak ; RSA, repeatedthreshold; HIE, 45 s of continuous cycling at 200% of the pretraining power at V 2 sprint ability; and , muscle biopsy.
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Table 1. The high-intensity training programme performed by the groups with either 1 (HIT-1) or 3 min (HIT-3) rest periods between intervals Training intensity Number of 2 (% pretraining (% pretraining O peak ) Week Session min intervals power at LTDmax ) power at V 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 6 7 8 8 9 8 8 10 9 8 10 9 8 7 6 140 140 140 150 150 150 160 160 160 170 170 170 170 170 170 92 92 92 98 98 98 105 105 105 111 111 111 111 111 111

one HIT-1 training session, separated by 1 week, in a counterbalanced order. Heart rate was averaged during each of the six completed intervals. Blood samples were taken from an earlobe at rest and 1 and 3 min following the sixth interval. To determine changes in MLa and PCr content and in [H+ ] following a typical HIT-1 and HIT3 training session, muscle samples (vastus lateralis) were also taken pre-exercise and after the sixth interval (1 min post-sixth interval for HIT-1 and 3 min post-sixth interval for HIT-3).
Capillary blood sampling

Subjects in the HIT-1 group performed an equal amount of total work (in kilojoules) during each session to their matched partner in the HIT-3 group. The only difference between the two groups was the rest period between intervals. Abbreviations: LTDmax , O peak , peak oxygen uptake. lactate threshold; and V 2

the interval-training programme. Based on our previous research (Edge et al. 2005, 2006a), the subjects performed between six and 10 intervals, each lasting 2 min in duration, three times per week (Monday, Wednesday and Friday) for ve consecutive weeks (Table 1). All training sessions were completed on mechanically braked cycle ergometers (818E; Monark, Stockholm, Sweden) and were preceded by a 5 min warm up at 50 W. Both intervaltraining groups were matched for training load and the duration of the work intervals. This was necessary to allow the comparison of the rest period per se on the training adaptations. The initial exercise intensity was 140% LTDmax , and this was increased by 10% at the end of each week for all subjects in order to maintain an intense training stimulus throughout the study (Table 1). The between-interval passive rest period was the only difference between the two training groups.
Physiological/metabolic differences between the two training programmes

Glass capillary tubes were used to collect 50 l of blood during the GXT (D957G-70-35; Clinitubes, Radiometer, Copenhagen, Denmark) and 100 l of blood during the HIE and training sessions (D957G70-125; Clinitubes, Radiometer). A hyperaemic ointment (Finalgon; Boehringer Ingelheim, Ingelheim, Germany) was applied to the earlobe 57 min prior to initial blood sampling, and all blood was sampled from the earlobe. Capillary blood samples were taken at rest and immediately following each 4 min stage of the GXT, and at rest and immediately after the HIE. Plasma pH and [BLa] were determined using a blood-gas analyser (ABL 625; Radiometer). The blood-gas analyser was regularly calibrated using precision standards and routinely assessed by external quality controls.
Muscle sampling and analysis

Five female university students not involved in the training study (recruited from the same population; 21 2 years O2 peak 49.5 3.2 ml kg1 min1 ) performed two old, V interval-training sessions to determine differences in heart rate, blood lactate concentration ([BLa]) and muscle metabolite accumulation between the two types of training. Each subject performed one HIT-3 and

On the day of the HIE-only cycle test, two incisions were made under local anaesthesia (5 ml, 1% Xylocaine) into the vastus lateralis of each subject. The rst incision was used for the pretest biopsy and then subsequently used for the post-test biopsy; pre- and post-test biopsy samples were taken with the needle inserted at different angles. The third biopsy sample was taken from the second site. All biopsies were performed with manual suction applied, and postbiopsy the whole needle was rapidly submerged in liquid nitrogen. The rst muscle sample was taken prior to warm up, during supine rest. The second muscle sample was taken immediately following the cessation of the HIE (<10 s), with the participant still seated on the cycle ergometer, while the third muscle sample was taken exactly 60 s after the removal of the needle used for the immediate postexercise muscle sample. This very short recovery period was chosen deliberately, because the rate of PCr resynthesis postexercise is rapid (Edge et al. 2006a) and because few studies have examined changes in the recovery of muscle metabolites over such a time frame. The subject remained on the cycle ergometer for all postexercise testing. Pre- and post-training samples were taken from opposite legs, at approximately the same position. The samples were then removed from the biopsy
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needle, rapidly frozen in liquid N2 and stored at 80 C until subsequent analysis.

Muscle [3 H]oubain binding sites Na+ ,K+ -ATPase content

and the supernatant was stored at 80 C. The contents of ATP, PCr and MLa were measured by bioluminescence (Arthur & Hochachka, 1995). Anaerobic ATP production was estimated by PCr + 1.5 MLa (Gore et al. 2001).

Muscle Na+ ,K+ -ATPase content was determined in quadruplicate using the vanadate-facilitated [3 H]oubain binding site content (Norgaard et al. 1984). Muscle samples were cut into 25 mg pieces and washed twice for 10 min in 37 C vanadate buffer containing 250 mM sucrose, 10 mM Tris, 3 mM MgSO4 and 1 mM NaVO4 (pH 7.27.4). Muscle samples were then incubated for 120 min at 37 C in the vanadate buffer with the addition of 3 H (106 M, 2.0 mCi ml1 ). After incubation, muscle samples were washed four times for 30 min in ice-cold vanadate buffer to remove any unbound [3 H]oubain, blotted on lter paper and weighed before being soaked overnight in vials containing 0.5 ml of 5% trichloroacetic acid and 0.1 mM ouabain. The following morning, 2.5 ml of scintillation cocktail (Opti-uor; Packard, Downers Grove, IL, USA.) was added before liquid scintillation counting of the 3 H activity. The content of [3 H]oubain binding site sites was calculated on the basis of the sample wet weight and the specic gravity of the incubation medium and samples and was expressed as picomoles per gram wet weight (Petersen et al. 2005).
Muscle carnosine content

Muscle homogenate pH

Freeze-dried, resting muscle samples (1.82.5 mg) were homogenized on ice for 2 min in a solution containing sodium uoride (10 mM) at a dilution of 30 mg of dry muscle per millilitre of homogenizing solution. The muscle homogenate was then placed in a circulating water bath at 37 C for 5 min prior to and during the measurement of pH. The pH measurements were made with a microelectrode (MI-415; Microelectrodes Inc., Bedford, NH, USA) connected to a pH meter (SA 520; Orion Research Inc., Cambridge, MA, USA).

Statistical analysis

For carnosine content, 12 mg of freeze-dried muscle was extracted with 20% w/v sulphosalicylic acid and further neutralized and diluted with 0.4 M borate buffer, pH 9.65. Extracts were analysed for carnosine by HPLC with a modication of the procedure described previously (Harris et al. 1990). The modication consisted of using a 25 mM Na2 HPO4 buffer, pH 6.8, instead of 12.5 mM acetate, pH 7.2, in order to improve peak separation. The method has a limit of detection for carnosine corresponding to 2 mol (g muscle dry weight)1 and a precision of approximately 3%. Due to insufcient muscle sample size, carnosine analysis was limited to four subjects for HIT-1 and three subjects for HIT-3.
Muscle ATP, PCr and MLa

All pre- and post-training data consist of six subjects per group, and all values are reported as means SD, unless otherwise stated. Two-way ANOVAs (HIT-1 and HIT-3 training groups), with repeated measures for time, were used to analyse data and to test for interaction and O2 peak , LT, muscle and main effects for measurements of V blood metabolites between the HIT-1 and HIT-3 groups. Signicance was accepted at P < 0.05 (SPSS 13.0; SPSS Inc., Chicago, IL, USA).

Results
Physiological responses to an acute training session

Freeze-dried (removed of blood and connective tissue) rest and postexercise muscle samples (23 mg) were enzymatically assayed for ATP, PCr and MLa content. The ATP, PCr and MLa were extracted from muscle samples by the addition of 6% perchloric acid, before being centrifuged (10,000g for 10 min). The supernatant was removed and neutralized by the addition of 2.4 mol l1 KOH and 3 mol l1 KCl. Samples were centrifuged again,
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Despite completing an identical amount of work, average heart rate during the six completed intervals was higher during the HIT-1 training session (181 10 beats min1 and 94% of maximal heart rate) than during the HIT-3 training session (175 9 beats min1 and 91% of maximal heart rate; P < 0.05). The HIT-1 training session also induced a greater peak postexercise [BLa] compared with the HIT-3 training session (15.5 3.0 versus 12.9 1.8 mmol l1 ; P < 0.05). Muscle [H+ ] (78.4 3.2 to 155 15 versus 79.2 2.6 to 125 8 nmol l1 ; P < 0.05) and MLa content (6.6 1.6 to 84.2 7.9 versus 7.2 2.1 to 46.9 3.1 mmol (g wet wt)1 ; P < 0.05) were both higher after HIT-1, while muscle PCr content (82.5 9.3 to 52.8 8.3 versus 83.1 8.6 to 63.4 9.8 mmol (g wet wt)1 ; P < 0.05) was less after HIT-1.

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O peak and lactate threshold before and Table 2. Body mass, V 2 after training for the groups with either 1 min (HIT-1) or 3 min (HIT-3) of rest between intervals Variable Body mass O peak V 2 (ml kg1 min1 ) Power at O peak (W) V 2 Lactate threshold (W) Group HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 Pretraining 62.1 58.2 45.6 45.6 217 202 142 132 7.7 4.7 6.8 4.4 34 21 26 17 Posttraining 62.1 58.9 50.0 49.6 242 221 153 152

Carnosine content

Change (%) 10 9 12 9 8 15

7.6 4.4 7.1 4.7 32 13 21 21

There were no signicant changes in resting muscle carnosine content after training for either the HIT-1 (from 28.4 5.0 to 31.6 6.6 mmol (kg dry wt)1 ) or the HIT3 group (from 30.9 21.5 to 36.4 10.0 mmol (kg dry wt)1 ).
Na+ ,K+ -ATPase content

Values are means SD; n = 6 per group. pretraining (P < 0.05).

Greater than

The muscle [3 H]ouabain binding site content was increased (from 22 to 26%; P < 0.05) after training for both the HIT-1 (191 48 versus 241 53 pmol (g wet wt)1 ) and HIT-3 groups (220 80 versus 270 106 pmol (g wet wt)1 ), with no differences between groups.
Changes in [BLa], pH and [HCO3 ] (measured during the HIE-only test)

Performance changes following training

O2 peak , power at V O2 peak There were signicant increases in V and LTDmax for both groups; however, there was no interaction effect for either of these variables (Table 2). Following training, the peak power increased during the rst sprint of the RSA test for both groups (10 versus 11%), with no differences between groups. Likewise, the total work of all ve sprints increased following HIT-1 (pre 252 29 to post 285 29 J kg1 ; +13%, P < 0.05) and HIT-3 (pre 241 15 to post 269 13 J kg1 ; +12%, P < 0.05), again with no differences between groups (Fig. 2).
Muscle metabolites (measured during the HIE-only test)

Blood lactate concentration, plasma pH and [HCO3 ], measured before and after the HIE-only test, pre- and post-training, are summarized in Table 4. There were no

Muscle metabolite data measured before, immediately after and 60 s after the HIE, pre- and post-training, are summarized in Table 3. There were no signicant changes in resting ATP, PCr, MLa or [H+ ] following either training programme (P > 0.05). From rest to postexercise, there were increases in MLa and [H+ ] and reductions in muscle ATP and PCr content, both before and after training. Despite performing the same amount of work during both the pretraining and post-training HIE tests, the immediate postexercise MLa content and [H+ ] were lower following training (P < 0.05); however, there were no differences between HIT-1 and HIT-3 for either of these variables (P > 0.05). Post-training there was no change in the recovery of MLa content or muscle pH at 60 s postexercise compared with pretraining. There was also no change in immediate postexercise ATP or PCr content following training. However, while there was no change in net ATP resynthesis, there was a faster PCr resynthesis rate following training for both HIT-1 (10.0 2.4 versus 20.2 5.2 mmol kg1 min1 ) and HIT-3 (9.3 1.6 versus 16.1 2.6 mmol kg1 min1 ).

Figure 2. Work (in joules per kilogram) completed during each of the ve sprints of the RSA test before and after the HIT-1 (A) and HIT-3 protocol (B) Each sprint duration was 6 s, followed by a 24 s passive recovery period. The RSA test was performed 60 s after the completion of the 45 s HIE test. Values are shown as means + SD. There were no signicant differences between groups pre- or post-training. Signicantly different from pretraining (P < 0.05); n = 6 for each group.
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Table 3. Muscle ATP, phosphocreatine, lactate (MLa), pH, calculated [H+ ] and estimated anaerobic ATP production (An-ATP) before, immediately after and 60 s after the HIE test, before and after 5 weeks of training Pretraining Variable ATP (mmol (kg dry wt)1 ) Phosphocreatine (mmol (kg dry wt)1 ) MLa (mmol (kg dry wt)1 ) pH [H+ ] (nmol l1 ) An-ATP (mmol (kg dry wt)1 ) Group HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 Before 20.1 20.5 84.0 81.2 7. 0 9.7 7.11 7.10 77.7 81.1 1.9 2.3 15.9 7.7 After 15.3 14.1 50.1 47.6 2.6 1.7 19.1 6.2 37.2 26.5 0.16 0.14 51.7 51.4 62 49 60 s post 18.8 15.7 62.2 61.5 57.3 53.3 6.98 6.94 106.2 120.3 2.0 2.9 16.4 7.1 31.9 32.7 0.11 0.13 29.5 38.4 Before 20.8 21.3 87.3 83.2 8.0 7.9 7.14 7.05 72.7 90.7 2.8 2.3 14.9 11.5 Post-training After 14.6 15.9 52.3 48.6 1.2 2.4 11.1 3.2 26.9 28.4 0.09 0.08 21.9 23.6 35 40 60 s post 18.6 20.6 74.5 67.2 47.4 39.6 7.07 6.96 87.2 112.1 3.5 1.5 5.4 6.7 19.1 23.6 0.09 0.11 17.1 30.3

1.8 69.1 2.4 70.2 0.02 6.87 0.05 6.81 3.0 144.2 7.1 160.7 132 131

3.5 54.5 3.2 53.7 0.04 7.01 0.06 6.92 6.5 99.6 13.5 122.6 111 107

Values are means SD. There were no signicant differences between groups pre- or post-training. An-ATP = Different from same time point pretraining (P < 0.05). Different from rest (P < 0.05).

PCr + 1.5 MLa.

signicant changes in resting or postexercise [BLa] or pH, but postexercise [HCO3 ] was higher following training.

3 min), which our pilot work demonstrated to increase the changes in muscle lactate, PCr and hydrogen ion concentration during representative training bouts.
The effects of training on metabolites

Discussion A clear nding from the present study was that highintensity interval training increased peak aerobic power, muscle PCr resynthesis postexercise and muscle Na+ ,K+ ATPase content, and enhanced the ability to recover from a bout of high-intensity exercise and perform repeated sprints. High-intensity interval training also resulted in smaller changes in muscle ATP, MLa and [H+ ] during a standardized high-intensity exercise bout, but did not alter their recovery postexercise. Importantly, we demonstrated for the rst time that these physiological and performance adaptations were not altered by decreasing the recovery period during our interval-training protocol (i.e. 1 versus

It is a common belief that exercise-induced changes in metabolites and ions are a crucial factor in the adaptation of the contracting muscle (Weston et al. 1997; Mohr et al. 2007). In the present study, we have assessed this hypothesis by employing two different training regimes, matched for total work, but which were associated with different changes in muscle lactate, PCr and hydrogen ion concentration. We show that reducing the length of the between-interval recovery period, thereby producing a greater decrease in PCr content during each training session (Spencer et al. 2006; Saraslanidis et al. 2011), did not affect post-training improvements in muscle

Table 4. Plasma pH, blood lactate ([BLa]) and plasma bicarbonate ion ([HCO3 ]) concentrations before and immediately after the HIE test, before and after 5 weeks of training Pretraining Variable pH [BLa] (mmol l1 ) [HCO3 ] (mmol l1 ) Group HIT-1 HIT-3 HIT-1 HIT-3 HIT-1 HIT-3 Pre-exercise 7.44 7.45 0.8 0.9 24.1 22.5 .02 .02 0.3 0.4 1.8 0.5 Postexercise 7.27 7.30 8.9 8.9 16.2 15.4 .03 .04 2.6 3.1 2.0 1.8 Post-training Pre-exercise 7.43 7.42 0.9 0.8 24.6 23.5 .02 .01 0.3 0.1 2.0 1.5 Postexercise 7.25 7.28 9.4 8.2 18.3 17.9 .02 .03 0.5 1.4 2.1 1.5

Values are means SD; n = 6 for each group. There were no signicant differences between groups pre- or post-training. Different from same time point pretraining (P < 0.05). Different from rest (P < 0.05).

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PCr resynthesis postexercise. Thus, contrary to our hypothesis, training sessions that were accompanied by greater decreases in muscle PCr content did not result in signicantly greater improvements in PCr resynthesis. It has also been suggested that a large increase in muscle [H+ ] during training may be a crucial factor in the adaptation of the muscle H+ -regulating systems (Weston et al. 1997). In contrast to our hypothesis, however, there was a similar decrease in immediate, postexercise MLa content and [H+ ] following training in both groups, despite a signicantly greater increase in muscle [H+ ] and MLa content during training with shorter rest periods (i.e. HIT-1). From our results, we cannot be sure of the contribution of a reduced anaerobic contribution (and improved aerobic power) and/or an improved H+ /MLa removal to the reduced accumulation of these ions. However, we show for the rst time that the length of the between-interval recovery period (hence changes in [H+ ] and MLa content during training) did not affect traininginduced changes in immediate, postexercise MLa content and [H+ ]. Furthermore, signicant differences in muscle [H+ ] and MLa content during training also did not affect training-induced changes in the removal of MLa or H+ 60 s postexercise. There were only small changes in muscle carnosine for both groups, suggesting that greater muscle buffering by carnosine was not the reason for the reduced H+ accumulation, and also suggesting that changes in muscle carnosine are not tightly coupled to the degree of H+ accumulation during training. The muscle carnosine levels of our women (30 mmol kg1 ) are higher than values previously reported for untrained women and men (20 mmol kg1 ; Mannion et al. 1992), but lower than that reported in well-trained male bodybuilders (40 mmol kg1 ; Tallon et al. 2005). The higher levels in our female participants compared with untrained men may be due to their history of training and competing in high-intensity sports. However, the bodybuilders had a much higher (33%) muscle carnosine content than our women, which may be due to differences in age (31 versus 18 years), length of intense training (13.7 versus 3 4 years of training) and the use of dietary supplements and anabolic steroids, which have been shown to affect carnosine levels (Penael et al. 2004). Long-term training appears to result in an elevation in muscle carnosine content (Parkhouse et al. 1985) and is associated with an increase in muscle buffering capacity (Parkhouse & McKenzie, 1984). However, the results of short-term training studies have been ambivalent (Harris et al. 2007; Hill et al. 2007), and we have shown that 5 weeks of interval training may not be long enough to increase muscle carnosine content. Furthermore, there were no differences in carnosine changes due to the rest period (hence the degree of H+ accumulation) during interval training.

Na+ ,K+ -ATPase content

Increased muscle Na+ ,K+ -ATPase content is consistently observed across different types of training (McKenna et al. 1993; Harmer et al. 2000). The increase in muscle Na+ ,K+ -ATPase content with training is thought to be vital in regulating muscle intracellular to extracellular [K+ ] gradients and thus preserving muscle excitability (McKenna & Hargreaves, 2008). Here we show that highintensity interval training by females resulted in a 22 26% increase in Na+ ,K+ -ATPase content. Increases in both training volume (Medb et al. 2001) and intensity (Evertsen et al. 1997), in already-trained participants, have previously been shown to improve Na+ ,K+ -ATPase content. We add new information to these prior ndings and show that, when training volume and intensity are matched, the length of the rest period between intervals during intense training does not affect changes in Na+ ,K+ ATPase content. Another interesting observation is that the Na+ ,K+ ATPase content was lower (20%) in our cohort than previously reported in men. This is in contrast to our earlier nding of no difference in Na+ ,K+ -ATPase content between recreational men and women (Murphy et al. 2007), but is consistent with a report that well-trained women have an 18% lower content of Na+ ,K+ -ATPase than well-trained men (Evertsen et al. 1997). We are unsure why the values of Na+ ,K+ -ATPase content were lower in the women of the present study. However, our post-training values were similar to those reported both in women (Murphy et al. 2007) and in untrained, recreationally trained and well-trained men (Green et al. 2004).
Recovery of repeated-sprint performance

It has previously been reported that a high aerobic tness and the ability to resynthesize PCr and to remove H+ are important determinants of single- and repeated-sprint performance (Bogdanis et al. 1996; Bishop et al. 2004; Spencer et al. 2006). In the present study, both groups had signicant improvements in the recovery of both single and repeated-sprint performance. It is possible that the reduced [H+ ] and greater PCr resynthesis 60 s after the HIE contributed to this improved performance. Additionally, the elevated Na+ ,K+ -ATPase content may have improved K+ regulation and contributed to improved sprint performance (McKenna et al. 1993; Mohr et al. 2007). However, while we show that repeated-sprint performance is improved following interval training, we also show that using short rest periods during highintensity interval training does not offer any advantage over the use of longer rest intervals when training intensity and volume are matched. This can most probably be attributed to the similar muscle adaptations induced by the two training programmes.
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Interval training and muscle metabolism

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Summary

Despite the absence of clear evidence, it has previously been hypothesized that exercise-induced changes in metabolites and ions are a crucial factor in the adaptation of the contracting muscle. Our results do not support this hypothesis, and show that altering the changes in muscle lactate, PCr and hydrogen ion concentration during training (via the manipulation of rest-period length between intervals) did not affect adaptations to skeletal muscle or aerobic power. Our results therefore suggest that the perturbation of muscle lactate, PCr and hydrogen ion concentration during high-intensity interval training is not a crucial factor regulating related adaptations of the contracting muscle when training intensity and volume are matched. However, further research is required because we employed high-intensity exercise training, and it is possible that the changes in both protocols exceeded a threshold required for adaptation. References
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