Overnight fasting compromises exercise intensity and volume during sprint interval training but improves high-intensity aerobic endurance

Abstract

Background:
The combined effects of sprint interval training (SIT) and exercising in the fasted state are unknown. We compared the effects of SIT with exogenous carbohydrate supplementation (SITCHO) and SIT following overnight fast (SITFast) on aerobic capacity (peak oxygen consumption: V̇ O2peak) and high-intensity aerobic endurance (time-to-exhaustion at 85% V̇ O2peak [T85%]).

Methods:
Twenty male cyclists were randomized to SITCHO and SITFast. Both groups performed 30-second all-out cycling followed by 4-minute active recovery 3 times per week for 4 weeks, with the number of sprint bouts progressing from 4 to 7. Peak power output (PPO) and total mechanical work were measured for each sprint interval bout. The SITCHO group performed exercise sessions following breakfast and consumed carbohydrate drink during exercise, whereas the SITFast group performed exercise sessions following overnight fast and consumed water during exercise. Before and after training, V̇ O2peak and T85% were assessed. Blood glucose, non-esterified fatty acids, insulin and glucagon concentrations were measured during T85%.

Results:
Overall PPO and mechanical work were lower in SITFast than SITCHO (3664.9 vs. 3871.7 Joules/kg; p=0.021 and 10.6 vs. 9.9 Watts/kg; p=0.010, respectively). Post- training V̇ O2peak did not differ between groups. Baseline-adjusted post-training T85% was longer in SITFast compared to SITCHO (19.7 ± 3.0 vs. 16.6 ± 3.0 minutes, ANCOVA p=0.038) despite no changes in circulating energy substrates or hormones.

Conclusions:
Our results suggest that SITFast compromises exercise intensity and volume but still can have a greater impact on the ability to sustain high-intensity aerobic endurance exercise compared to SITCHO.

Full text

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Overnight fasting compromises exercise intensity and volume during sprint interval
training but improves high-intensity aerobic endurance
Tasuku Terada1, Saeed R Toghi Eshghi2, Yilina Liubaoerjijin2, Michael Kennedy2, Etienne
Myette-Côté3, Kevin Fletcher2, Normand G. Boulé2*.
1Faculty of Rehabilitation Medicine, Department of Occupational Therapy, University of Alberta,
Edmonton, AB Canada T6G 2G4; 2Faculty of Physical Education and Recreation, 1-02 Li Ka
Shing Centre for Health Research Innovation, Edmonton, AB Canada T6G 2E1; 3School of
Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC Canada
V1V 1V7
Running title: Fasted vs. fed sprint interval training (SIT)
Address for correspondence: Dr. Normand G. Boulé, University of Alberta, Faculty of
Physical Education and Recreation, 1-02 Li Ka Shing Centre for Health Research Innovation
Edmonton, AB Canada, T6G 2E1; Phone: (780) 492- 4695; E-mail: nboule@ualberta.ca
Word count: 3501

2
ABSTRACT
BACKGROUND: The combined effects of sprint interval training (SIT) and exercising in the
fasted state are unknown. We compared the effects of SIT with exogenous carbohydrate
supplementation (SITCHO) and SIT following overnight fast (SITFast) on aerobic capacity (peak
oxygen consumption: V
̇O2peak) and high-intensity aerobic endurance (time-to-exhaustion at 85%
V
̇O2peak [T85%]). METHODS: Twenty male cyclists were randomized to SITCHO and SITFast.
Both groups performed 30-second all-out cycling followed by 4-minute active recovery 3 times
per week for 4 weeks, with the number of sprint bouts progressing from 4 to 7. Peak power
output (PPO) and total mechanical work were measured for each sprint interval bout. The SITCHO
group performed exercise sessions following breakfast and consumed carbohydrate drink during
exercise, whereas the SITFast group performed exercise sessions following overnight fast and
consumed water during exercise. Before and after training, V
̇O2peak and T85% were assessed.
Blood glucose, non-esterified fatty acids, insulin and glucagon concentrations were measured
during T85%. RESULTS: Overall PPO and mechanical work were lower in SITFast than SITCHO
(3664.9 vs. 3871.7 Joules/kg; p=0.021 and 10.6 vs. 9.9 Watts/kg; p=0.010, respectively). Post-
training V
̇O2peak did not differ between groups. Baseline-adjusted post-training T85% was longer
in SITFast compared to SITCHO (19.7 ± 3.0 vs. 16.6 ± 3.0 minutes, ANCOVA p=0.038) despite no
changes in circulating energy substrates or hormones. CONCLUSIONS: Our results suggest that
SITFast compromises exercise intensity and volume but still can have a greater impact on the
ability to sustain high-intensity aerobic endurance exercise compared to SITCHO.

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Key words: exercise, aerobic power, submaximal performance, Wingate, time to fatigue,
carbohydrate supplementation
Introduction
Aerobic capacity (i.e., peak oxygen consumption; V
̇O2peak) and high-intensity aerobic endurance
(i.e., ability to sustain work at intensities above 85% V
̇O2peak) constitute fundamental aspects of
performance associated with endurance sports.1 Thus, training strategies to improve these
parameters continue to be an important area of research in sport science. Traditionally,
continuous aerobic endurance training with aerobic intervals (>4 minutes in length) has been
used to increase aerobic capacity and endurance in trained athletes.2-4 However, anaerobic sprint
interval training (SIT) of shorter duration (30 seconds) has recently emerged and adopted as an
alternative strategy to induce rapid muscular aerobic adaptations, such as increased muscle
glycogen content, oxidative enzyme expression, and mitochondrial biogenesis.5,6 SIT
supplemented into regular training has been shown to increase V
̇O2peak in well-trained athletes7
and high-intensity aerobic endurance in recreationally active individuals.8,9
Modification to nutritional availability has also been proposed as a potent strategy to
modulate exercise training responses. It has been well established that carbohydrate
supplementation can acutely increase aerobic cycling endurance.10 However, training with
reduced carbohydrate availability have advantages in enhancing glycogen content11-13 and
activating signaling pathways12 that lead to the activation of aerobic enzymes14 to a greater extent
than the same exercise performed with ample carbohydrate supplementation. Aerobic exercise
training under restricted exogenous carbohydrate availability has been shown to improve
supramaximal cycling capacity15 and time trial performance16 to a greater extent than the same
training performed with high carbohydrate availability. Thus, while exercise with increased

4
carbohydrate availability can have positive immediate effects on aerobic performance and/or
recovery, high carbohydrate availability during exercise training may hamper some of the
important adaptive responses induced by exercise training.
To date, the effects of SIT and fasted-state exercise have been studied independently.
While both types of training appear to share overlapping mechanisms for muscular adaptations
(e.g., increased muscular glycogen content and aerobic enzymatic activities), it is unknown if the
combination of SIT and fasted-state exercise training (SITFast) can induce greater improvements
in aerobic capacity and high-intensity aerobic endurance compared to SIT performed under
ample carbohydrate condition (SITCHO). The primary purpose of the study was to compare the
effects of SITCHO and SITFast on aerobic capacity and high-intensity aerobic endurance. The
secondary purpose was to compare the effects of SITCHO and SITFast on circulating substrates and
hormone concentrations involved in energy metabolism during high-intensity aerobic endurance
cycling. We hypothesized that SITFast would induce greater increases in aerobic capacity and
high-intensity endurance compared to SITCHO.
Material and methods
Participants
Male cyclists were recruited through advertisement. The inclusion criteria for the study were: 1)
cycle more than 3 hours per week; 2) non-smokers; 3) not currently performing fasted-state
training; 4) between 18 and 45 years of age; and 5) able to train in the morning 3 days per week.
We recruited participants who cycle more than 3 hours per week to minimize changes in cycling
economy over the exercise training period. Potential participants completed a questionnaire to
verify their eligibility. Physical Activity Readiness Questionnaire (PAR-Q)17 was also completed
to screen for any contraindications to performing high-intensity exercise. Participants gave

5
written informed consent prior to participation. All experimental procedures performed were in
accordance with Declaration of Helsinki and were approved by University of Alberta Health
Research Ethics Board.
Experimental design
A randomized parallel group trial was conducted. After screening for eligibility, baseline aerobic
capacity (V
̇O2peak) was assessed, followed by high-intensity aerobic endurance cycling (time-to-
exhaustion at 85% V
̇O2peak [T85%]) on a subsequent day at least 48 hours apart. Participants were
then randomized to either SITCHO or SITFast by a researcher not involved in the study. Without
being blinded to the conditions, both groups performed 3 training sessions per week with 48-72
hours between each session for 4 weeks (e.g., training on Monday, Wednesday, and Friday
morning). After successful completion of 4-week SIT, V
̇O2peak and T85% tests were repeated
using the same protocols used in baseline testing. The post-training T85% took place
approximately 72 hours following the last day of SIT. The post-training assessment of V
̇O2peak
occurred on a subsequent day, at least 48 hours apart. Overview of experimental protocol is
summarized in Figure 1. All exercise testing and sessions were completed on a stationary bike
ergometer (Monark, Ergomedic 894E, Varberg, Sweden).
Training protocols
All participants trained 3 days per week for 4 weeks in controlled laboratory conditions. Training
sessions were interspersed with at least 48 hours of recovery. The progressive SIT training
included 30 seconds of all-out cycling efforts18 with a resistance equivalent to 0.075 kg x body
mass (kg), separated by 4 minutes of unloaded pedaling. Participants performed 4 bouts per

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session in week 1, 5 bouts per session in week 2, 6 bouts per session in week 3, and 7 bouts per
session in week 4 (Figure 1). Before each high-intensity bout, an instruction was given to
increase pedaling speed as high as possible, and the appropriate load was applied promptly.
Participants were given verbal encouragement to maintain maximal pedaling speed throughout
the 30-second period.
Participants in the SITCHO group completed 3-day dietary record (breakfast only) before
participating in SIT. If breakfast carbohydrate intake was <2.5g/body mass (kg),19 participants
were instructed to supplement carbohydrate intake by adding cereal and/or orange juice to their
breakfast. Breakfast was consumed at least 1 hour prior to each training session to minimize
gastrointestinal distress. During each training session, 591 mL Gatorade (0 g fat, 35g
carbohydrate, and 0 g protein; Pepsico Beverages Canada, 5205 Satellite Drive Mississauga, ON,
Canada) was also provided to the SITCHO group. The SITFast group reported to the laboratory
after ≥10 hours of overnight fast, and consumed only water during exercise sessions.
To examine if restricted exogenous carbohydrate availability compromised training
volume and power output (PO), we measured peak power output (PPO), mean PO, and fatigue
index during each sprint interval bout. Reflective makers were placed on the flywheel, and a
photo cell interfaced to a computer determined the number of flywheel revolutions during a
sprint interval bout using a custom designed software program. PO was calculated as the product
of resistance and flywheel revolutions every 0.1 second. PPO was determined from the first 5
seconds,20,21 and fatigue index was computed from the difference between 5-second PPO and the
PO during the last 5 seconds of the test divided by the PPO multiplied by 100.18 Mechanical
work of each sprint interval bout was quantified for each participant by multiplying the mean PO
by total sprint time (i.e., work = Joules/seconds x 30 seconds). Weekly training volume was

7
calculated by summing mechanical work of all sprint intervals performed in the week, which was
adjusted for participants’ body mass to allow between-group comparison. PPO was also adjusted
for different body mass.
Aerobic capacity (V
̇O2peak) test
Participants performed an incremental test to exhaustion on a cycle ergometer at a self-selected
cadence to determine V
̇O2peak and VT using a TrueMax® (ParvoMedics, Sandy, UT) metabolic
measurement system. First and second VT (VT1 and VT2) were determined as previously
described22 by a researcher blinded to conditions. The initial 3 stages of the incremental test were
2 minutes each, and the weight applied to the flywheel were 1.0, 1.5 and 2.0 kg, respectively.
Resistance was increased by adding 0.3 kg per minute thereafter until volitional exhaustion.
V
̇O2peak was determined as the highest value averaged over 20 seconds before reaching volitional
exhaustion. The cadence, saddle and handle bar positions used for the baseline test were recorded
and reproduced in subsequent tests.
High-intensity aerobic endurance (T85%) test
Endurance performance was defined as the length of time that cycling could be maintained at an
intensity requiring 85% of VO2peak. Participants reported to the laboratory after an overnight fast
(≥10 hours) and were provided with standardized breakfast: a granola bar (8 g fat, 28 g
carbohydrate, and 3 g protein; Nature Valley granola bar, General Mills, Mississauga, ON,
Canada) and meal replacement drink (6 g fat, 32 g carbohydrate, and 9 g protein; Ensure regular,
Abbott, Saint-Laurent, QC, Canada). Participants were given 30 minutes of rest, during which a
catheter was inserted in the antecubital vein. The intravenous line was kept patent with saline

8
solution flushes. Participants then performed 5-minute warm-up at pedaling resistance
corresponding to 25% of resistance prescribed for the T85% test. The T85% test was performed at
the same cadence selected for the V
̇O2peak test while adjusting workload to match the intensity at
85% of individually determined V
̇O2peak. Participants were blinded to the elapse of time and
remained seated for the entire testing duration. No verbal encouragement was provided. The test
was terminated when cadence fell below the predetermined speed for more than 5 seconds and
followed by 5 minutes cool-down.
Blood samples were collected into a 10-mL EDTA vacutainer tube (Becton, Dickson and
Company, ON, Canada) immediately before warm-up, every 10 minutes during the test, and
immediately following cool-down. Respiratory gases were analysed using metabolic
measurement system between 6th and 9th minutes every 10 minutes to determine the rates of
oxygen consumption (V
̇O2) and carbon dioxide production (V
̇CO2). Rating of perceived
exhaustion (RPE) was recorded immediately before the respiratory gas collection period using
the Borg 6-20 RPE scale.23. Heart rate was also recorded at this time.
Biochemical analyses
Two mL of collected blood samples were transferred into a tube with 6.7 µL aprotinin (Millipore,
MA, USA), and 0.25 mL were transferred into a tube containing 1.0 mL ice-cold 8% perchloric
acid. Aprotinin was added to inhibit proteases known to interfere with the determination of
glucagon concentration, while perchloric acid was added to deproteinize the sample. Tubes were
centrifuged and acquired plasma was cooled and stored in a -80 °C freezer until analysis. NEFA
(Wako Diagnostics, CA, USA), plasma glucose, and lactate concentrations were determined
enzymatically with spectrophotometric assays. Glucagon and insulin concentrations were

9
measured using a Multi-Spot® Assay System with a Sector® Imager 2400 (Meso Scale
Discovery®, MD, USA). All assays were run in duplicate and mean values were used for
subsequent analyses. The intra-assay coefficient of variation for glucagon, insulin, NEFA,
plasma glucose, and lactate were 6.5%, 4.2%, 5.6%, 4.4%, and 4.0%, respectively.
Statistical analysis
At baseline, independent t-tests were used to compare participant characteristics between SITCHO
and SITFast. While differences did not reach statistical significance, relative V
̇O2peak and T85% in
SITFast were lower compared to SITCHO at baseline (53.1 ± 7.8 vs. 57.3 ± 7.8; p=0.248 and 12.6 ±
8.0 vs. 17.4 ± 7.5; p=0.184, respectively). Typically, poor baseline measures lead to greater
improvements.24 We therefore performed analysis of covariance (ANCOVA) on post-training
measures with baseline measures included as covariates to determine if training outcomes
differed between SITCHO and SITFast.25 These analyses are presented as adjusted means (also
known as least square means) throughout the article. For circulating substrates and hormones, we
separately performed ANCOVA on measures obtained before exercise, during exercise, and after
exercise.
For the analysis of fatigue index, mechanical work and PPO adjusted for body mass,
weekly measures were compared between SITCHO versus SITFast by using repeated measure
ANOVA. Data are expressed as mean ± standard deviation (SD). Normality of residual
distributions was examined with Shapiro-Wilk test. Insulin concentration was log-transformed to
achieve normality. Data were analysed using IBM SPSS Statistics for Windows, version 23
(IBM Corp., Armonk, NY, USA).

10
Results
Participants
Twenty-five participants completed the baseline tests. Of these, 5 withdrew from the study. Two
participants randomized to SITCHO withdrew due to schedule incompatibility. Three participants,
1 from SITCHO and 2 from SITFast withdrew from the study due to severe dizziness and nausea
following the first SIT session. As a result, 9 participants in the SITCHO group and 11 participants
in the SITFast group were included in the analyses. All participants completed 12 exercise training
sessions, except for 1 in SITFast who missed 1 session in the 4th week. There were no significant
differences between SITCHO and SITFast in baseline characteristics, V
̇O2peak, T85%, VT1 or VT2
(Table 1).
Training volume, PPO, and fatigue index
Weekly mean mechanical work and PPO adjusted for body mass, and fatigue index for both
groups are summarized in Figure 2. Repeated measure ANOVA showed that, with an addition of
a sprint interval bout per week, total mechanical work increased every week in both groups
(p<0.001). Overall mechanical work adjusted for body mass was significantly higher in SITCHO
compared to SITFast (3871.7 vs. 3664.9 Joules/kg; p=0.021, Cohen’s d=0.29). Similarly, overall
PPO adjusted for body mass was significantly higher in SITCHO compared to SITFast (10.6 vs. 9.9
Watts/kg; p=0.010, Cohen’s d=0.57). Fatigue index did not differ between the groups.
Aerobic capacity (V
̇O2peak) and VT
There was no difference in post-training V
̇O2peak between SITCHO and SITFast (Figure 3). Pre and
adjusted post-training V
̇O2 at VT1 were 33.7 ± 9.2 vs. 33.7 ± 5.1 mL·O2/kg/min for SITCHO and

11
30.1 ± 8.2 vs. 33.1 ± 5.9 mL·O2/kg/min for SITFast, respectively. Pre and adjusted post-training
V
̇O2 at VT2 were 46.3 ± 9.0 vs. 43.8 ± 7.2 mL·O2/kg/min for SITCHO and 42.3 ± 9.0 vs. 43.2 ±
6.7 mL·O2/kg/min for SITFast, respectively. There were no differences in post-training VT1 or
VT2 between groups.
High-intensity aerobic endurance (T85%)
Oxygen consumption expressed as a percentage of baseline V
̇O2peak was 87.8 ± 6.0% and 86.2 ±
4.0% during pre- and post-T85%, respectively, in SITCHO. In SITFast, pre and post %V
̇O2peak were
86.4 ± 7.7% and 89.7 ± 8.4%, respectively. Mean PO for SITCHO and SITFast during T85% were
261.8 ± 23.4 and 261.8 ± 23.4 Watts, respectively. There was no significant difference between
groups.
Adjusted mean post-training T85% was significantly longer in SITFast than SITCHO (19.7 ±
3.0 min vs. 16.6 ± 3.0 min, p=0.038, effect size estimated by partial Eta squared=0.230; Figure
4). The rate of perceived exertion, heart rate, or respiratory exchange ratio (RER) did not change
or differ between groups (Table 2). We only analysed RER during the first 10 minutes because
second metabolic cart measures (i.e., between 16-19 minutes) were only available from 6
participants. In addition, because 2 participants reached volitional exhaustion before the first
metabolic cart measurement, these participants were not included in the RER analysis.
Blood sample analyses
The results of blood sample analyses are summarized in Table 2. At baseline, there were no
significant differences in plasma glucose, lactate, NEFA, insulin or glucagon concentrations
before, during, and immediately after T85% tests between SITCHO and SITFast. However, adjusted
glucose and glucagon concentrations measured immediately before the post-training T85% test

12
were significantly lower in SITFast compared to SITCHO (4.2 ± 0.8 vs. 5.1 ± 0.3 mmol/L, p=0.024,
partial Eta squared=0.28 and 29.5 ± 12.7 vs. 53.2 ± 25.7 pmol/L, p=0.022, partial Eta
squared=0.30). Immediately following the post-training T85% test, adjusted glucose
concentrations were lower in SITFast than SITCHO (5.1 ± 1.1 vs. 6.0 ± 1.0 mmol/L, p=0.011,
partial Eta squared=0.34).
Discussion
To the best of our knowledge, this is the first study to compare the effects of sprint interval
training performed under both limited and ample exogenous carbohydrate conditions on aerobic
capacity and high-intensity aerobic endurance. By manipulating exogenous carbohydrate
availability during training, we showed that SIT performed after an overnight fast leads to better
high-intensity aerobic endurance performance compared to SIT performed with additional
carbohydrate intake after adjusting for baseline differences. This may have training implications
for athletes since the ability to sustain power outputs at high intensity (i.e., >85% V
̇O2peak) has
been suggested as a critical determinant factor of success in endurance events.19,26 Interestingly,
the SITFast group increased T85% more than the SITCHO group despite lower body mass-adjusted
mean mechanical work and PPO during training.
Previous studies have shown as little as 6 sessions of SIT over 2 weeks are a potent
stimulus to enhance skeletal muscle buffering capacity, oxidative capacity, and glycogen content
to a similar degree as 90 to 120 minutes of endurance exercise training5 and double time-to-
exhaustion at 80% V
̇O2peak27 in healthy individuals. Our results indicated that performance of
experienced cyclists can be enhanced using very similar protocols with a potential of added
performance benefit by training under fasted state. Exercise intensity in our study during the

13
T85% test was ~88% V
̇O2peak, which corresponds to the exercise intensity used in a previous study
investigating high-intensity aerobic endurance performance in well-trained competitive cyclists.1
Despite similar exercise intensity relative to individually-determined V
̇O2peak, cycling duration of
the current study were shorter than the previous study (i.e., ~15 minutes at baseline vs. ~45
minutes), possibly due to lower overall training status or aerobic capacity (55.3 ± 7.5 vs. 67.3 ±
3.9 ml·O2/kg/min) in our participants. Despite shorter time-to-exhaustion, our results translate
into an improved capacity to cover additional 1.1 ± 1.7 and 2.1 ± 1.4 km at given exercise
intensity in SITCHO and SITFast groups, respectively. The mean difference of an extra kilometre
performed in the SITFast strengthens the potential benefit of fasted state training on cycling
performance.
Our results showing increased high-intensity aerobic endurance despite no changes in
V
̇O2peak is similar to previous studies in well-trained runners and cyclists.28,29 Considering
previously reported central adaptation (i.e., increased left ventricular mass and stroke volume)
and increased V
̇O2peak in response to higher volume SIT (5 sessions per week for 8 weeks),30 we
suspect that our SIT volume was not sufficient to induce changes in V
̇O2peak. Alternatively,
because changes in V
̇O2peak have predominantly been observed in untrained participants,6,31-35 a
greater training stimulus may be required to induce physiological adaptation in more fit
participants such as ours. Likewise, while a previous study showed increases in VT1 and VT2 in
response to SIT,7 there was no group level changes in threshold in our study in either SITCHO or
SITFast. This discrepancy may also be attributable to lower training volume in our study (i.e., 66
SIT bouts vs. 96 SIT bouts). We found no correlations between the baseline V
̇O2peak and changes
in T85%.

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At baseline, there were no differences in the concentrations of circulating hormones and
energy substrates. However, after training, glucose concentrations measured immediately before
and after T85% were significantly lower in SITFast compared to SITCHO. Additionally, glucagon
concentration measured immediately before post-training T85% was also lower in SITFast
compared to SITCHO. We do not have clear explanation on why glucagon concentration was
lower in SITFast than SITCHO following training. However, others have demonstrated that
antecedent episode of increased cortisol concentration36 can blunt counter regulatory responses,
including glucagon, in response to hypoglycaemia on a subsequent day. Thus, repeated episodes
of augmented cortisol concentration induced by fasted-state exercise37 may have suppressed
glucagon concentration following SITFast. Alternatively, higher energy demands on the liver
during SITFast may have increased AMP to ATP ratio in the liver. This could have stimulated
hepatic 5’ AMP-activated protein kinase (AMPK) activity, thereby increasing glucagon
sensitivity in the liver38 and reducing glucagon concentrations. This lower glucagon
concentration is likely to be a contributing factor for lower glucose concentration in SITFast
before post-training T85%.
As acutely manipulating substrate availability can exert profound effects on muscle
energy stores and patterns of fuel metabolism during exercise, when repeated over time such
interventions can have the potential to modulate numerous adaptive processes in skeletal
muscle.39 Our results of longer aerobic cycling endurance following SITFast than SITCHO without
changes in energy substrates mobilization or oxidation rates suggest that the change was due to
unmeasured parameters. While we did not perform muscle biopsy, available evidence has shown
that commencing exercise with limited exogenous carbohydrate intake13,40 or withholding
glucose intake during exercise40,41 increases muscle AMPK activity, which stimulates

15
downstream regulators, such as peroxisome proliferator-activated receptor-γ 1α (PGC-1α),42,43 a
protein known to facilitate mitochondrial biogenesis.44-47 Additionally, compared to the same
exercise training under a fed condition, the fasted-state exercise facilitates glycogenolysis,48 and
thereby increases glycogen content to a greater extent once replenished.11,49,50 Altogether, given
that both SIT and fasted-state exercise can enhance oxidative enzyme activities and glycogen
contents, we speculate that the effects of SIT and fasted-state exercise may be additive and
resulted in a greater degree of improvement in T85%.
A limitation of our study includes relatively large heterogeneity in baseline V
̇O2peak and
T85%. We performed ANCOVA to adjust for these baseline differences instead of analysing
percent changes as percent changes are generally more pronounced for participants with poorer
baseline measures. Nonetheless, ANCOVA does not eliminate the possibility that differing
physiological and/or biochemical responses occurred as a result of different starting points.
Additionally, while we instructed participants not to alter their dietary intake, we did not capture
macronutrient contents of their dietary intake during the training period. Thus, the potential
influence of residual confounding arising from different dietary intake cannot be ignored. Lastly,
we were unable to provide mechanisms underpinning performance changes. Further study is
warranted to determine the specific mechanisms that lead to better adaptation.
Conclusion
This study provides some novel information for sport scientists and coaches involved in the
training of athletes. We have demonstrated that performing sprint interval training in a fasted
state enhances actual performance to a greater extent than the same training performed under an
ample carbohydrate condition. These results could be considered a novel ergogenic aid that has

16
the potential to elevate both team and individual sport performance. Future replication of these
results will affirm the understanding of how energy status can influence adaptation in training
programs.
Acknowledgments: This study was supported by grants from the Sport Science Association of
Alberta (SSAA) and University of Alberta, Faculty of Physical Education and Recreation, the
Human Performance Funding Award (HPF). The funding sources had no role in design, conduct
or reporting of the study. The authors would like to thank Irina Smith and Ryan St James for
their support with data collection. The authors would also like to thank Dr. Craig Steinback for
his help with participant randomization.
Duality of interest: The authors declare that there is no duality of interest associated with this
manuscript.
Contribution statement: TT, MK, EMC and NGB were responsible for the conception and
design of the study. Collection and assembly of data was carried out by TT, SRTE, YL, MK,
EMC, KF and NGB. Analysis and interpretation of data was carried out by TT, SRTE, YL, and
NGB. TT drafted the article and SRTE, YL, MK, EMC, KF, and NGB critically revised the
article for important intellectual content. TT, SRTE, MK, EMC and NGB obtained funding. All
authors gave final approval for the article to be published.

17
References:
1. Coyle E, Coggan A, Hopper M, Walters T. Determinants of endurance in well-trained
cyclists. J Appl Physiol 1988;64:2622-2630
2. Neal CM, Hunter AM, Brennan L, O'Sullivan A, Hamilton DL, DeVito G, et al. Six
weeks of a polarized training-intensity distribution leads to greater physiological and
performance adaptations than a threshold model in trained cyclists. J Appl Physiol
2013;114: 461-471
3. Lindsay F, Hawley J, Myburgh K, Schomer H, Noakes T, Dennis S. Improved athletic
performance in highly trained cyclists after interval training. Med Sci Sports Exerc,
1996;28:1427-1434
4. Seiler K, Kjerland G. Quantifying training intensity distribution in elite endurance
athletes: Is there evidence for an "optimal" distribution? Scand J Med Sci Sports
2006;16:49-56
5. Gibala MJ, Little JP, van Essen M, Wilkin GP, Burgomaster KA., Safdar A, et al. Short-
term sprint interval versus traditional endurance training: Similar initial adaptations in
human skeletal muscle and exercise performance. J Physiol 2006;575:901-911
6. Burgomaster KA, Howarth KR, Phillips SM, Rakobowchuk M, MacDonald MJ, McGee
SL, Gibala MJ. Similar metabolic adaptations during exercise after low volume sprint
interval and traditional endurance training in humans. J Physiol 2008;586:151-160
7. Laursen P, Shing C, Peake J, Coombes J, Jenkins D. Influence of high-intensity interval
training on adaptations in well-trained cyclists. J Strength Cond Res 2005;19:527-533

18
8. Harmer A, McKenna M, Sutton J, Snow R, Ruell P, Booth J, et al. Skeletal muscle
metabolic and ionic adaptations during intense exercise following sprint training in
humans. J Appl Physiol 2000;89:1793-1803
9. Hazell TJ, MacPherson REK, Gravelle BMR, Lemon PWR. 10 or 30-S sprint interval
training bouts enhance both aerobic and anaerobic performance. Eur J Sport Sci
2010;110:153-160
10. Yaspelkis B, Patterson J, Anderla P, Ding Z, Ivy J. Carbohydrate supplementation spares
muscle glycogen during variable-intensity exercise. J Appl Physiol 1993;75:1477-1485
11. Stannard SR, Buckley AJ, Edge JA, Thompson MW. Adaptations to skeletal muscle with
endurance exercise training in the acutely fed versus overnight-fasted state. J Sci Med
Sport 2010;13:465-469
12. Van Proeyen K, Szlufcik K, Nielens H, Ramaekers M, Hespel P. Beneficial metabolic
adaptations due to endurance exercise training in the fasted state. J Appl Physiol
2011;110:236-245
13. De Bock K, Richter EA, Russell AP, Eijnde BO, Derave W, Ramaekers M, et al.
Exercise in the fasted state facilitates fibre type-specific intramyocellular lipid breakdown
and stimulates glycogen resynthesis in humans. J Physiol 2005;564:649-660
14. Morton JP, Croft L, Bartlett JD, MacLaren DPM, Reilly T, Evans L, et al. Reduced
carbohydrate availability does not modulate training-induced heat shock protein
adaptations but does upregulate oxidative enzyme activity in human skeletal muscle. J
Appl Physiol 2009;106:1513-1521

19
15. Marquet L, Brisswalter J, Louis J, Tiollier E, Burke LM, Hawley JA, et al. Enhanced
endurance performance by periodization of carbohydrate intake: "Sleep low" strategy.
Med Sci Sports Exerc 2016;48:663-672
16. Cochran AJR, Myslik F, Macinnis MJ, Percival ME, Bishop D, Tarnopolsky MA, et al.
Manipulating carbohydrate availability between twice-daily sessions of high-intensity
interval training over 2 weeks improves time-trial performance. Int J Sport Nutr Exerc
Metab 2015;25: 463-470
17. Warburton D, Jamnik V, Bredin S, Gledhill N. The physical activity readiness
questionnaire (PAR-Q+) and electrical phsyical activity readiness medical examination
(eRAPmed-X+). Health & Fitness Journal of Canada 2011
18. Baror O. The wingate anaerobic test - an update on methodology, reliability and validity.
Sports Med 1987;4:381-394
19. Burke LM. Fueling strategies to optimize performance: Training high or training low?
Scand J Med Sci Sports 2010;20:48-58
20. Forbes SC, Kennedy MD, Boule NB, Bell G. Determination of the optimal load setting
for arm crank anaerobic testing in men and women. Int J Sports Med 2014;35:835-839
21. Mandic S, Quinney H, Bell G. Modification of the wingate anaerobic power test for
rowing: Optimization of the resistance setting. Int J Sports Med 2004;25:409-414
22. Lucia A, Hoyos J, Perez M, Chicharro J. Heart rate and performance parameters in elite
cyclists: A longitudinal study. Med Sci Sports Exerc 2000;32:1777-1782
23. Borg GAV. Psychophysical bases of perceived exertion. Med Sci Sports Exerc
1982;14:377-381

20
24. Vickers A, Altman D. Statistics notes - analysing controlled trials with baseline and
follow up measurements. BMJ 2001;323:1123-1124
25. Vickers AJ. The use of percentage change from baseline as an outcome in a controlled
trial is statistically inefficient: A simulation study. BMC Med Res Methodol 2001;1:6
26. Joyner MJ, Coyle EF. Endurance exercise performance: The physiology of champions. J
Physiol 2008;586:35-44
27. Burgomaster KA, Hughes SC, Heigenhauser GJF, Bradwell SN, Gibala MJ. Six sessions
of sprint interval training increases muscle oxidative potential and cycle endurance
capacity in humans. J Appl Physiol 2005;98:1985-1990
28. Bangsbo J, Gunnarsson TP, Wendell J, Nybo L, Thomassen M. Reduced volume and
increased training intensity elevate muscle Na+-K+ pump alpha2-subunit expression as
well as short- and long-term work capacity in humans. J Appl Physiol 2009; 107:1771-
1780
29. Laursen P, Blanchard M, Jenkins D. Acute high-intensity interval training improves T-
vent and peak power output in highly trained males. Can J Appl Physiol 2002;27:336-348
30. Matsuo T, Saotome K, Seino S, Shimojo N, Matsushita A, Iemitsu M, et al. Effects of a
low-volume aerobic-type interval exercise on VO2max and cardiac mass. Med Sci Sports
Exerc 2014;46:42-50
31. Willoughby TN, Thomas MPL, Schmale MS, Copeland JL, Hazell TJ. Four weeks of
running sprint interval training improves cardiorespiratory fitness in young and middle-
aged adults. J Sports Sci 2016;34:1207-1214

21
32. Macpherson REK, Hazell TJ, Olver TD, Paterson DH, Lemon PWR. Run sprint interval
training improves aerobic performance but not maximal cardiac output. Med Sci Sports
Exerc 2011;43:115-122
33. Sandvei M, Jeppesen PB, Stoen L, Litleskare S, Johansen E, Stensrud T, et al. Sprint
interval running increases insulin sensitivity in young healthy subjects. Arch Physiol
Biochem 2012;118:139-147
34. Barnett C, Carey M, Proietto J, Cerin E, Febbraio MA, Jenkins D. Muscle metabolism
during sprint exercise in man: Influence of sprint training. J Sci Med Sport 2004;7:314-
322
35. Trilk JL, Singhal A, Bigelman KA, Cureton KJ. Effect of sprint interval training on
circulatory function during exercise in sedentary, overweight/obese women. Eur J Appl
Physiol 2011;111:1591-1597
36. Davis S, Shavers C, Costa F, Mosqueda-Garcia R. Role of cortisol in the pathogenesis of
deficient counterregulation after antecedent hypoglycemia in normal humans. J Clin
Invest 1996;98:680-691
37. Galbo H, Holst J, Christensen N. Effect of different diets and of insulin on the hormonal
response to prolonged exercise. Acta Physiol Scand 1979;107:19-32
38. Trefts E, Williams AS, Wasserman DH. Exercise and the regulation of hepatic
metabolism. Prog Mol Biol Transl Sci 2015;135:203-25
39. Hawley JA. Nutritional strategies to modulate the adaptive response to endurance training.
Nestle Nutr Inst Workshop Ser 2013;75:1-14

22
40. Akerstrom TCA, Birk JB, Klein DK, Erikstrup C, Plomgaard P, Pedersen BK, et al. Oral
glucose ingestion attenuates exercise-induced activation of 5 '-AMP-activated protein
kinase in human skeletal muscle. Biochem Biophys Res Commun 2006;342:949-955
41. Civitarese AE, Hesselink MKC, Russell AP, Ravussin E, Schrauwen P. Glucose
ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat
oxidative genes. Am J Physiol Endocrinol Metab 2005; 289:E1023-E1029
42. Bartlett JD, Hawley JA, Morton JP. Carbohydrate availability and exercise training
adaptation: Too much of a good thing? Eur J Sport Sci 2015;153-12
43. Margolis LM, Pasiakos SM. Optimizing intramuscular adaptations to aerobic exercise:
Effects of carbohydrate restriction and protein supplementation on mitochondrial
biogenesis. Adv Nutr 2013;4:657-664
44. Jager S, Handschin C, St-Pierre J, Spiegelman BM. AMP-activated protein kinase
(AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc Natl
Acad Sci USA 2007;104:12017-12022
45. Zong HH, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, et al. AMP kinase is
required for mitochondrial biogenesis in skeletal muscle in response to chronic energy
deprivation. Proc Natl Acad Sci USA 2002;99:15983-15987
46. Hardie DG. Energy sensing by the AMP-activated protein kinase and its effects on
muscle metabolism. Proc Nutr Soc 2011;70:92-99
47. Gibala M. Molecular responses to high-intensity interval exercise. Appl Physiol Nutr
Metab 2009;34:428-432

23
48. De Bock K, Derave W, Ramaekers M, Richter EA, Hespel P. Fiber type-specific muscle
glycogen sparing due to carbohydrate intake before and during exercise. J Appl Physiol
2007;102:183-188
49. Van Proeyen K, Szlufcik K, Nielens H, Pelgrim K, Deldicque L, Hesselink M, et al.
Training in the fasted state improves glucose tolerance during fat-rich diet. J Physiol
2010;588:4289-4302
50. Nybo L, Pedersen K, Christensen B, Aagaard P, Brandt N, Kiens B. Impact of
carbohydrate supplementation during endurance training on glycogen storage and
performance. Acta Physiol 2009;197:117-127

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Table titles
Table 1. Baseline demographic and aerobic characteristics
Table 2. Pre- and post-training substrate oxidation, energy substrate concentration and hormone
concentrations before, during and after time-to-exhaustion at a workload corresponding to 85%
of baseline V
̇O2peak (T85%)
Figure legends
Figure 1. Schematic presentation of study flow.
SIT: sprint interval training
SITCHO: sprint interval training with breakfast prior to training and carbohydrate during exercise;
SITFast: sprint interval training following an overnight fast. V
̇O2peak: peak oxygen consumption;
T85%: time-to-exhaustion test at 85% V
̇O2peak.
*The same protocols as baseline tests were used.
Figure 2. a) Mechanical work, b) peak power output, and c) fatigue index over the 4-week
training period.
SITCHO: sprint interval training performed under ample carbohydrate; SITFast: sprint interval
training following an overnight fast. Mechanical work is sum of mechanical work during SIT
only. Peak power output was calculated as the sum of peak power output over number of SIT
performed. *Repeated measures ANOVA showed a significant difference between SITCHO and
SITFast (main effects: p=0.021 and p=0.010, respectively). asignficantly different from week 1;
bsignficantly different from week 1 and 2; csignficantly different from week 1,2, and 3 (all
p<0.001).

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Figure 3. Peak oxygen consumption in response to SITFast and SITCHO.
SITCHO: sprint interval training with breakfast prior to training and carbohydrate during exercise;
SITFast: sprint interval training following an overnight fast. V
̇O2peak: peak oxygen consumption
Post-training V
̇O2peak was adjusted for baseline differences.
Figure 4. High-intensity aerobic endurance in response to SITFast and SITCHO.
*significantly different from SITCHO (ANCOVA adjusting for baseline values, p=0.038).
SITCHO: sprint interval training with breakfast prior to training and carbohydrate during exercise;
SITFast: sprint interval training following an overnight fast. T85%: time-to-exhaustion test at 85%
peak oxygen consumption. Post-training T85% was adjusted for baseline differences.

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Table 1. Baseline demographic and aerobic characteristics
SITFast, n=11
SITCHO, n=9
t-test
Mean ± SD
Mean ± SD
p-value
Age, years
33.3 ± 7.2
34.0 ± 8.2
0.837
Height, cm
180.4 ± 7.9
177.7 ± 6.6
0.416
Weight, kg
81.1 ± 13.3
78.9 ± 11.7
0.701
BMI, kg/m2
24.8 ± 2.7
25.0 ± 3.1
0.899
V
̇O2peak, L·O2/min
4.3 ± 0.6
4.5 ± 0.4
0.419
V
̇O2peak, mL·O2/kg/min
53.1 ± 7.8
57.3 ± 7.8
0.248
VT1, %V
̇O2peak
55.5 ± 8.7
58.0 ± 10.8
0.577
VT2, %V
̇O2peak
78.1 ± 8.6
80.0 ± 7.2
0.597
T85%, min
12.6 ± 8.0
17.4 ± 7.5
0.184
BMI: body mass index; V
̇O2peak: peak oxygen consumption; VT1: first ventilatory
threshold; VT2; second ventilatory threshold; SITCHO: sprint interval training with
ample carbohydrate; SITFast: sprint interval training after overnight fast; T85%: Time-to-
exhaustion at 85% of individually determined V
̇O2peak.

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Table 2. Pre- and post-training substrate oxidation, energy substrate concentration and hormone concentrations before, during and
after time-to-exhaustion at a workload corresponding to 85% of baseline V
̇O2peak (T85%)
SITFast, n=11
SITCHO, n=9
Before T85%
During T85%
After T85%
Before T85%
During T85%
After T85%
RER
pre
0.99 ± 0.04
0 .98 ± 0.03
post
0.99 ± 0.03
0.99 ± 0.04
RPE
pre
14 ± 2
14 ± 2
post
14 ± 1
13 ± 1
Heart rate (bpm)
pre
176 ± 9
171 ± 12
post
175 ± 7
175 ± 14
Glucose, mmol/L
pre
5.3 ± 0.9
4.1 ± 0.4
4.8 ± 1.0
4.5 ± 0.9
3.9 ± 1.5
5.1 ± 0.9
post
4.2 ± 0.8*
3.8 ± 0.6
5.1 ± 1.1*
5.1 ± 0.3
3.8 ± 0.7
6.0 ± 1.0
Lactate, mmol/L
pre
1.1 ± 0.5
8.9 ± 2.4
8.8 ± 2.2
1.4 ± 0.5
7.2 ± 3.0
6.9 ± 1.9
post
1.3 ± 0.5
8.0 ± 2.5
7.8 ± 2.1
1.2 ± 0.5
7.5 ± 2.6
8.7 ± 2.0
NEFA, mmol/L
pre
0.24 ± 0.11
0.12 ± 0.05
0.17 ± 0.12
0.21 ± 0.12
0.11 ± 0.03
0.11 ± 0.03
post
0.19 ± 0.11
0.13 ± 0.05
0.20 ± 0.10
0.12 ± 0.08
0.10 ± 0.03
0.10 ± 0.03

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Insulin, pmol/L
pre
777 ± 1069
612 ± 622
580 ± 324
868 ± 572
438 ± 327
979 ± 1025
post
698 ± 509
545 ± 686
254 ± 161
763 ± 909
1159 ± 914
391 ± 176
Glucagon, pmol/L
pre
49.3 ± 20.4
32.7 ± 14.2
44.3 ± 20.3
40.7 ± 11.8
44.6 ± 28.9
42.8 ± 14.4
post
29.5 ± 12.7*
49.7 ± 11.6
46.4 ± 22.9
53.2 ± 25.7
40.9 ± 23.7
41.2 ± 11.7
RER: respiratory exchange ratio; CHO: carbohydrate; RPE: rate of perceived exertion; NEFA: non-esterified fatty acids; SITCHO:
sprint interval training with ample carbohydrate; SITFast: sprint interval training after overnight fast; Before T85%: blood sample was
collected immediately before exercise following 5 minutes of sitting on cycle ergometer without pedaling; During T85%:calorimetry
measures were taken during the 6-9 minute interval and blood sample was collected at minute 10; After T85%: blood sample was
collected immediately after cool-down. Adjusted means are presented for post-training values. *Significantly different from SITCHO
(p<0.05). For circulating substrates and hormones during T85%, n=6 for SITFast and n=8 for SITCHO. For RER and RPE during T85%,
n=9 for SITFast and n=9 for SITCHO.

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