Muhmmed M.Atkn ,Ysemin Güzel ,Süleymn Bulut ,Şükrn N.Ko¸sr ,Glenn K.McConell ,Hüseyin H.Turngöl ,*

a Division of Nutrition and Metabolism in Exercise,Faculty of Sport Sciences,Hacettepe University,Ankara 06690,Turkey

b Institute for Health and Sport,Victoria University,Melbourne,VIA 3011,Australia

Abstract Background:High-intensity interval training (HIIT) induces similar or even superior adaptations compared to continuous endurance training.Indeed,just 6 HIIT sessions over 2 weeks signif icantly improves maximal oxygen uptake (VO2max),submaximal exercise fat oxidation,and endurance performance.Whether even faster adaptations can be achieved with HIIT is not known.Thus,we aimed to determine whether 2 sessions of HIIT per day,separated by 3 h,every other day for 5 days(double HIIT(HIIT-D),n=15)could increase VO2max,submaximal exercise fat oxidation,and endurance capacity as effectively as 6 sessions of HIIT over 2 weeks(single HIIT(HIIT-S),n=13).Methods:Each training session consisted of 10×60 s of cycling at 100%of VO2max interspersed with 75 s of low-intensity cycling at 60 watt(W).Pre-and post-training assessments included VO2max,time to exhaustion at～80%of VO2max,and 60-min cycling trials at～67%of VO2max.Results:Similar increases(p ＜0.05)in VO2max(HIIT-D:7.7%vs.HIIT-S:6.0%,p ＞0.05)and endurance capacity(HIIT-D:80.1%vs.HIIT-S:79.2%,p ＞0.05) were observed.Submaximal exercise carbohydrate oxidation was reduced in the 2 groups after exercise training (HIIT-D:9.2%,p=0.014 vs.HIIT-S:18.8%,p=0.012)while submaximal exercise fat oxidation was signif icantly increased in HIIT-D(15.5%,p=0.048)but not in HIIT-S(9.3%,p=0.290).Conclusion:Six HIIT sessions over 5 days was as effective in increasing VO2max and endurance capacity and was more effective in improving submaximal exercise fat oxidation than 6 HIIT sessions over 2 weeks.

Keywords:Double interval training;Exercise metabolism;Performance;Substrate oxidation

1.Introduction

The World Health Organization recommends at least 150 min of moderate-intensity physical activity per week or at least 75 min of vigorous-intensity physical activity per week for healthy adults.1Unfortunately,over a quarter of all adults,approximately 1.4 billion people,do not reach these recommended physical activity levels.2Given that a lack of time is the most commonly cited barrier to exercise adherence,recent studies have focused on exercise training models that can be effectively undertaken in a short period of time.There is a growing body of research suggesting that high-intensity interval training(HIIT)is such a time-saving exercise model.3

HIIT refers to an intermittent style of exercise training involving short bursts of high-intensity exercise,for example,intermittent exercise that achieves ≥90% of maximal heart rate(HR)and maximal oxygen uptake(VO2max),separated by periods of recovery or rest.4This exercise model has been ranked number 1 in the health and fitness trends in 2018 due to its time-saving nature.5Although HIIT has been used by athletes in their training since the beginning of the 20th century,its use has recently gained momentum6and the number of published studies relevant to HIIT has increased exponentially over the past decade.4

Research indicates that an HIIT training program ranging from 2 weeks to 8 months in length is a time-eff icient exercise strategy to confer wide-ranging health benef its,including changes in body composition and decreases in visceral adiposity.7－11HIIT also increases cardiorespiratory fitness,exercise performance,7－9,12and fat oxidation during sub-maximal exercise.11The underlying mechanisms behind these improvements may include,at least partly,increases in skeletal muscle mitochondrial biogenesis and mitochondrial function,which manifests itself as greater oxidative phosphorylation capacity.12The briefest time that HIIT has been used thus far,as reported in published research studies,has been 2 weeks,with 6 HIIT sessions over that time,and these studies found increases in VO2max,fat oxidation during sub-maximal exercise and endurance capacity,as well as skeletal muscle metabolic adaptations.8－11It is also worth noting that some studies in the published literature have found that physiological adaptations took place when a non-HIIT program involved even less than 2 weeks of exercise training.For example,Green et al.13found increased levels of skeletal muscle glucose and lactate transporters after 3 consecutive days of cycling at 60%of VO2max.Phillips et al.,14who reported on the shortest regimen of training that produced exercise-induced improvements in substrate oxidation,also documented an increased rate of intramuscular lipolysis and a decreased rate of glycogen oxidation after 2 h cycling at 60%of peak oxygen uptake(VO2peak)daily over 5 days.To our knowledge,no study has examined whether these types of benef icial effects can be achieved when HIIT is used over a time frame of less than 2 weeks.It has been found that 2 sessions of HIIT per day increases cardiorespiratory fitness15and cycling time trial performance,12most likely because HIIT increases skeletal muscle oxidative phosphorylation capacity and markers of exercise-induced mitochondrial biogenesis.16This may be because when exercise training is done while muscle glycogen is depleted(e.g.,when exercise has taken place earlier on the same day),greater adaptations occur than when the same type and level of exercise is undertaken when muscle glycogen is at normal levels.17Indeed,HIIT conducted with reduced carbohydrate (CHO)availability increases whole-body fat oxidation during a steady-state ride and time trial performance compared to when HIIT is conducted with normal CHO availability.17,18Similarly,Hansen and colleagues19found that knee extensor exercise conducted twice per day every second day for 10 weeks resulted in increased resting muscle glycogen levels and citrate synthase activity compared with training only once per day for 10 weeks.Hansen et al.19hypothesized that this was because the lower muscle glycogen content available for the second bout of exercise on the same day acted as a greater stimulus for exercise adaptations.Furthermore,HIIT interventions comprising total 60 min of intense cycling over 5 days may result in health benef its that are similar or superior to the benef its derived from 2 weeks of HIIT,especially as it relates to the effects on VO2max,a powerful independent predictor of cardiovascular morbidity and mortality.20Conf irming that 5 days of HIIT intervention is as effective as 2 weeks of HIIT intervention will enable a new HIIT model to be developed that takes advantage of the time-saving nature of HIIT for those who want to experience both health and athletic benef its of training over a shorter period of time.Thus,we examined whether twice-daily HIIT sessions conducted every other day over 5 days improves VO2max,fat oxidation during submaximal exercise,and time to exhaustion (TTE) in a way that is similar to once-daily HIIT conducted every other day(on weekdays) over 2 weeks.We hypothesized that the 6 HIIT sessions performed over 5 days would provide these health benef its at a level that was similar or superior to once-daily HIIT sessions conducted every other day on weekdays over 2 weeks.

2.Methods

2.1.Participants

Twenty-eight healthy recreationally active males (age:25± 4 years;height:176.3 ± 6.0 cm;weight:72.7 ± 8.1 kg;mean ± SD) who participated in exercise (e.g.,cycling,running,soccer) 2－3 times per week volunteered for the study.Participants were randomly assigned to either the double high-intensity interval group (HIIT-D) (n=15) or the single high-intensity interval group (HIIT-S) (n=13).Inclusion criteria were:recreationally active males,non-smokers,aged 20－30 years,who did not engage in any systematic training program,including endurance,sprint,and resistance.Participants were excluded if they had a known exercise-limiting cardiovascular,respiratory or metabolic illness or were taking any medication or supplements known to affect metabolism.Participants were informed of the benef its and risks of the investigation prior to signing an institutionally approved informed written consent document for participation in the study.Participants also completed the International Physical Activity Questionnaire prior to their participation.The Hacettepe University Clinical Researches Ethics Boards approved the study,and the study conformed to the Declaration of Helsinki.

2.2.Experimental approach to the problem

The experimental protocol included a familiarization procedure,pre-tests,6 HIIT sessions,and post-tests.The pre-tests consisted of,in the following order,on separate days,with at least 48 h between each test,(a) VO2maxtest,(b) steady-state sub-maximal exercise test,and (c) TTE test (Fig.1).The post-tests,which were identical to the pre-tests,were then conducted beginning 72 h after the last HIIT training session(Fig.1).The study duration,inclusive of pre-and post-training testing,was 3 weeks for the HIIT-D group and 4 weeks for the HIIT-S group.During the HIIT phase,the HIIT-D group performed 2 consecutive training sessions on the same day,separated by a rest period of 3 h,during which participants were provided with a meal containing 10 kcal/kg composed of 58% CHO,27% fat,and 15% protein.The participants were asked to consume the meal provided during the first hour of the rest time;they were allowed water ad libitum.The HIIT-S group participated in the HIIT sessions on weekdays once per day (3 times/week) for 2 weeks.The HIIT-D and the HIIT-S groups were matched for total work.The participants were required to refrain from any strenuous physical activity for the 48 h before the pre-and post-tests,and from alcohol and any exercise for 24 h before each training day.To avoid performance fluctuations because of circadian rhythms,the first daily exercise sessions of individuals in the HIIT-D group and all exercise sessions of the HIIT-S group were performed in the morning (9:00 a.m.－noon),while individuals in the HIIT-D group completed their second daily sessions in the afternoon(noon－3:00 p.m.)

Fig.1.Study design.Fri=Friday;HIIT=high-intensity interval training;Mon=Monday;SS=steady-state submaximal test;T=training;Tue=Tuesday;TTE=time to exhaustion;VO2max=maximal oxygen uptake;Wed=Wednesday.

2.3.Procedures

2.3.1.Familiarization procedures

At least 2 weeks prior to baseline measurements,all participants performed familiarization trials to become oriented with all the testing procedures.The familiarization tests consisted of VO2max,the TTE test,and an HIIT session involving 10 × 60 s of exercise at VO2maxinterspersed by 75 s rest at 60 watt (W).These familiarization tests were separated by 24 h.

2.3.2.Reproducibility of exercise performance tests

Twelve individuals(age:27.1±3.2 years;height:180.3±3.1 cm;weight:70.1 ± 4.4 kg) who weren’t participants in our main study performed 2 VO2maxtests and 2 TTE tests on separate days at least 1 week apart in order to gauge the reproducibility of the exercise performance tests.The coeff icient of variation for the VO2maxtest and TTE test was 1.3%and 9.2%,respectively,which is similar to that reported in other studies.9The intraclass correlation coeff icient (ICC),calculated using the equation of Weir,21was 0.91 and 0.95 for VO2maxand TTE,respectively.The SE of the measurement,calculated by SE of the measurement=was 0.16 and 1.89 for VO2maxand TTE,respectively,where the SD is the SD of the scores from all participants.

2.3.3.VO2maxtests

The participants in our study performed an incremental exercise test to exhaustion on a cycle ergometer (Monark Ergodemic 894 E a;Pike Bike,Varberg,Sweden)to determine VO2max.The initial 3 stages of the test consisted of 2 min of cycling at 60 W,120 W,and 150 W,respectively,and then the workload was increased by 30 W every minute until volitional exhaustion.HR was recorded continuously during the test using an HR monitor (Heart Rate Monitor;Garmin International Inc.,Olathe,KS,USA).Breath-by-breath expired air measurements were performed throughout the test using an on-line gas analysis system (Quark CPET;COSMED Cardio-Pulmonary Exercise Testing,Rome,Italy).Prior to each test,O2,CO2,and volume calibrations were made according to procedures prescribed by the manufacturer.The value used for VO2maxcorresponded to the highest value achieved over a 30-s sampling period.Peak power output was calculated according to the following formula:Peak power output=Wcom+(t/60)×30,where Wcomis the last completed power output W,t is the time completed in the final unf inished workload (in seconds),60 is the increment duration(s) in each workload and 30 is the workload increment.22The test was considered valid when at least two of the following criteria were met:(a) plateauing of VO2while increasing work rate (increase of no more than 2 mL/kg/min),23(b)a respiratory exchange ratio(RER)greater than 1.1,and(c)an HR within 10 beats/min of the predicted maximum(220－age(beat/min)).24

2.3.4.Steady-state submaximal exercise test

The participants cycled for 60 min on an ergometer at a workload calculated to elicit～67% of pre-training VO2maxat a cadence of 60 repetition per minute (rpm) before and after the 6 sessions of HIIT.Three to 4 h before the pre-training and post-training 60-min ride,the participants consumed a 10-kcal/kg standard meal composed of 58% CHO,27% fat,and 15%protein as recommended by the Institute of Medicine of National Academies25(a normal eating pattern should derive from about 55%－65% CHO,30% fat,and 10%－20%protein).The volume of water consumed during the test was recorded and then replicated during the post-training test.Ratings of perceived exertion(RPE)were recorded every 15 min during exercise.Respiratory gases were collected at 13－18 min,28－33 min,43－48 min,and 55－60 min during exercise in order to measure VO2and CO2production and volume of RER.11RER was calculated as the ratio between CO2production and VO2,while fat oxidation(FOX)and CHO oxidation(CHOX)were calculated using stoichiometric equations,26with the assumption that the urinary nitrogen excretion rate was negligible:

2.3.5.TTE test

The effect of HIIT intervention on endurance capacity was assessed as TTE.After a 3-min warm-up at 60 W,participants cycled to volitional exhaustion on a cycle ergometer at a workload eliciting～80%of VO2max(at 80 rpm).No temporal,verbal or physiological feedback was applied during the test.9The test was terminated when the pedal cadence dropped below 75 rpm for more than 5 s or when the participants voluntarily stopped cycling.Exercise duration and distance covered were recorded.Expired breath samples for the determination of ventilation volume,O2uptake,CO2production,and RER were collected in the 6－10-min period after the TTE began.9HR was monitored continuously throughout the test.

2.3.6.HIIT

Seventy-two hours following the TTE test,participants started the exercise training protocol,which consisted of either 6 HIIT sessions over 5 days for the HIIT-D group or 6 sessions over 2 weeks for the HIIT-S group (Fig.1).To determine the most feasible protocol for the study design(HIIT administered twice daily),we conducted a pre-study involving different HIIT and sprint interval training protocols,including the allout Wingate test,10×4-min cycling at 85%－90% of VO2maxand 10×60 s cycling at 100%of VO2max,interspersed by 75 s of cycling at 60 W.Following the tests,the recruited participants(n=12;age:25.6± 3.4 years) were asked if they could do the same exercise within 3 h.Based on the responses from the participants,we decided that the 10×60 s protocol was feasible for our training model.The protocol consisted of repeated 60 s efforts of high-intensity cycling at a workload that corresponded to the peak power output reached by the participants at the end of the VO2maxtest (264 ± 48 W).These intervals were interspersed by 75 s of cycling at low intensity (60 W)for recovery.10Each session was started and finished with a 3-min warm-up/warm-down at 60 W.All participants completed 10 high-intensity intervals during each training session.

2.3.7.Nutritional monitoring

To minimize any diet-induced variability in post-tests,food diaries were given to each participant to record food and fluid consumption for the 24 h prior to each pre-test,and the participants were asked to replicate this during post-training testing.The diaries were photocopied and given back to the participants,and a dietician contacted them regularly to ensure fidelity.Household units such as cups,pieces,or plates were used to express portion size.Ingredients of mixed dishes were specif ied.Serving sizes were calculated by product name and standard weights of food items.All the results were calculated and analyzed by the same experienced dietician using BeB˙IS 6.1 software (Nutrition Information System,Dr J.Erhardt,Stuttgart,Hohenheim,Germany).

2.3.8.Statistical analyses

The sample size was calculated using the statistical program G*Power software (Version 3.1.9.5.;Institut der Universit¨at Bonn,Bonn,Germany),27which revealed that 13 participants per group were needed,with an effect size of 0.25,an α of 0.05,and a power of 0.85 as reported by Schubert et al.28Cohen’s effect sizes(d)were classif ied as small(0.20),moderate(0.60),and large(0.80)29and were calculated from data on the means,the number of participants,and the pooled SDs for each trial.A two-way repeated measures analysis of variance(time×group) was used to determine whether main effects existed between the groups and over time,followed by a Bonferroni post hoc test.Independent sample t tests were also used to determine if differences between groups existed at baseline across outcome variables.Statistical analyses were computed using SPSS Statistics for Windows,Version 21.0(IBM Corp.,Armonk,NY,USA),and the level of signif icance was set at p ＜0.05.All data are presented as mean±SD.

3.Results

The characteristics of the participants are presented in Table 1.There were no differences (p ＞0.05) in age,anthropometric measures,VO2max,peak power output,or maximal HR between the 2 groups at baseline.The mean percentage of maximal HR for overall HIIT sessions was 91.5% ± 1.0% in the HIIT-S group and 90.8% ± 1.6% in the HIIT-D group(p ＞0.05).Dietary analyses revealed no differences in thetotal energy or macronutrient content of diets before exercise training compared to after exercisetraining(Table 2).

Table 1Participant characteristics at baseline(mean±SD).

3.1.VO2max

VO2max(mL/kg/min)increased by 6.1% ±5.9% (41.8±6.9 mL/kg/min to 44.5±6.0 mL/kg/min,p=0.001)in HIIT-S and 7.7%±4.1%(45.2±6.6 mL/kg/min to 48.7±6.1 mL/kg/min;p＜0.001)in HIIT-D(d=0.68),with no signif icant difference between the 2 exercise protocols(Fig.2A).Peak power output elicited by the participants at the end of the VO2maxtest also increased (p＜0.001;d=0.09) to a similar extent in both exercise protocols(by 13.4%±4.7%,259.8±26.8 W to 294.9±24.6 W in the HIIT-Sgroup and by 10.4%±4.4%,264.9±50.2 W to 292.5±53.3 W in the HIIT-D group;Fig.2B).Both groups showed similar increases(p＞0.05)in maximal expired ventilation(VE)with the HIIT training(HIIT-S:120.6±25.1 L/min vs.145.4±22.1 L/min;HIIT-D:131.7±30.2 L/min vs.156.6±30.1 L/min,p＜0.001;d=0.43).

3.2.Steady-state submaximal exercise and substrate oxidation

Given that exercise training increased VO2maxin both groups and the absolute workload was the same before and after the training,the percentage of pre-training VO2maxwas lower (p＜0.05;Table 3)after exercise training in both groups,with no difference between groups(HIIT-S:decreased by 10.7%from 72.4%to 61.7%;HIIT-D decreased by 6.4%from 67.2%to 60.8% pre-training VO2max;d=0.13).The RER during the steady-state submaximal exercise test wassignif icantly lower at 15 min in the HIIT-Sgroup(d=0.01)and at 30 min(d=0.33)and 45 min(d=0.28)in the HIIT-D group(p＜0.05).Lowering of VE was signif icant at 15 min(d=0.26),30 min (d=0.04),and 60 min (d=0.01)in the HIIT-Sgroup and at 30 min(d=0.22)and 60 min(d=0.01)in the HIIT-D group(p＜0.05),with no differences between groups(p＞0.05).There was a signif icant decrease in absolute oxygen uptake during 60 min of cycling at 15 min (d=0.57)and 60 min (d=0.35)after training in the HIIT-Sgroup(p＜0.05),yet no signif icant change in the HIIT-D group was observed (p＞0.05).The relative percentage of the VO2maxat 15 min(d=0.15),30 min(d=0.13),45 min(d=0.26),and 60 min (d=0.20) during the steady-state submaximal exercise test was signif icantly decreased after training in both groups (p ＜0.05),while no signif icant difference between groups was present (main effect for condition,p ＞0.05;Table 3).

Fig.2.Changes in VO2max and peak power output pre-and post-intervention for the HIIT-S and HIIT-D groups.* p ＜0.05,signif icant difference within group.Values are means and individual changes.HIIT-D=double high-intensity interval training;HIIT-S=single high-intensity interval training;VO2max=maximal oxygen uptake;W=watt.

Table2Nutritional data preceding the pre-and post-testsfor HIIT-Sand HIIT-D groups(mean±SD).

Table 3Effects of 2 HIIT regimes on VO2,RER,and VE during steady-state submaximal exercise test(mean±SD).

Total fat oxidation during submaximal exercise increased by 15.5% in HIIT-D (19.6 ± 7.5 g to 22.6 ± 7.1 g;15.4%;p=0.048),with a non-signif icant increase in HIIT-S (18.6 ±6.8 g to 20.4 ± 6.8 g;9.2%;p=0.290;d=0.32).Total CHO oxidation signif icantly decreased by 18.8%(132.6±33.5 g to 107.6±30.7 g;p=0.014)and 9.2%(118.4±18.6 g to 107.4± 23.2 g;p=0.012;d=0.01) in the HIIT-S group and in the HIIT-D group,respectively,with no difference between groups(main effect for condition,p ＞0.05).

There was also a reciprocal decrease in CHO oxidation at 15 min (d=0.12),30 min (d=0.03),45 min (d=0.11),and 60 min (d=0.02) after training in the HIIT-S group and at 30 min (d=0.03) and 60 min (d=0.02) in the HIIT-D group(p ＜0.05;Figs.3A and 3B).Fat oxidation increased at 15 min in the HIIT-S group (p ＜0.05;d=0.36;Fig.3C) and at 30 min in the HIIT-D group (p ＜0.05;d=0.39;Fig.3D).HR was signif icantly lower after training at 15 min(d=0.48),30 min(d=0.31),45 min (d=0.27),and 60 min (d=0.35) of cycling at～67% of pre-training VO2maxin both groups (p ＜0.05;Figs.3E and 3F),with no differences between groups.

3.3.TTE

TTE increased by 79.2% in the HIIT-S group (13.2 ±5.1 min vs.23.8±10.7 min,p ＜0.003;d=1.2)and 80.1%in the HIIT-D group (17.6 ± 6.9 min vs.31.7 ± 17.9 min,p ＜0.001;d=0.56;Fig.4),with no difference between groups.

Oxygen uptake during exercise was not affected by exercise training in either group (p ＞0.05).There was also a similar(p ＞0.05)signif icant decrease (p ＜0.005)for both groups in HR recorded from the 6th to 10th min of the TTE tests(d=0.39).Also,VE was lower in both groups after training(HIIT-S:114.1 ± 14.7 L/min vs.102.5 ± 14.1 L/min;p=0.008;HIIT-D group:105.4±20.9 L/min vs.99.7±19.2 L/min;p=0.026;d=0.16),with no differences between the groups.The average absolute VO2during the TTE test did not differ (p ＞0.05) between groups.During the 6th to 10th min of the TTE tests,the HIIT-S group reached 97.0% of pre-training VO2maxand the HIIT-D group reached 92.2%of pre-training VO2max.The same power outputs represented 94.3% and 88.4% of pre-training VO2maxin the HIIT-S and HIIT-D groups,respectively,during the TTE exercise test at the same absolute workload post-training,which is similar for both groups when compared with pre-training(p ＞0.05).

Fig.3.Changes in carbohydrate oxidation (A) in the HIIT-S group and (B) in the HIIT-D group;fat oxidation (C) in the HIIT-S group and (D) in the HIIT-D group;and heart rate(E)in the HIIT-S group and(F)in the HIIT-D group,during steady-state submaximal exercise test at pre-and post-intervention for the HIIT-S group and HIIT-D group.Values are presented as mean±SD.*p ＜0.05,signif icant difference within group.

4.Discussion

The benef icial effects of HIIT on health,body composition,skeletal muscle oxidative capacity,and exercise metabolism have been well documented.4,30Prior to our study,the shortest HIIT study consisted of 6 sessions of HIIT over 2 weeks,with 1 session of HIIT conducted 3 days per week.8－11,31The main findings of this study indicated that only 5 days of HIIT with 2 sessions conducted every other day resulted in improvements in VO2max,submaximal exercise fat and CHO oxidation and cycle endurance capacity that were similar to those achieved with 6 HIIT sessions conducted over 2 weeks.Ours is the shortest HIIT study that has shown such improvements.

4.1.HIIT and VO2max

We found that 6 sessions of HIIT completed in 5 days resulted in increases in VO2maxsimilar in magnitude to those that were observed for 6 sessions of HIIT administered over 2 weeks.8－11It should be noted that although HIIT was conducted over 5 days in our study,the post-training VO2maxtest was conducted 72 h after the last HIIT session,or 7 days after the first HIIT session.Therefore,even though there were 5 days of HIIT training,there were 8 days during which adaptations to exercise could manifest themselves.

Fig.4.Changes in time to exhaustion at pre-and post-intervention for the HIIT-S and HIIT-D groups.Values are means and individual changes.*p ＜0.05,signif icant within group.HIIT-D=double high-intensity interval training;HIIT-S=single high-intensity interval training.

We are aware of very few studies using any type of exercise that have examined whether a 5-day duration of exercise training increases VO2max.One group of researchers found that 2 h of cycling at～60%of VO2peakover 3－4 consecutive days did not increase VO2peak,32but the same group found that 10 consecutive days of such exercise increased VO2maxby 4.3%.33A study by Hickson et al.34is the only study that we are aware of that reported an increase in VO2maxas quickly as our HIIT-D protocol did.Hickson et al.34found that 6 consecutive days of strenuous endurance exercise consisting of six 5-min cycling intervals corresponded to the increases seen in VO2maxwhen participants cycled for 3 days and ran as far as they could for 40 min over 3 days,which increased VO2maxby 5%.

It is quite remarkable that VO2maxincreases under these circumstances after about a week of exercise training.One would not expect that there would be time for increases given the classic limiting factors for determining VO2max,specif ically,maximum cardiac output.7,35Indeed,even 2 weeks of HIIT8or 6 weeks of sprint interval training36does not increase maximum cardiac output despite increasing VO2max,as pointed out in a recently published review.37

Although VO2maxis usually considered to be limited by maximum cardiac output,35it is possible that skeletal muscle mitochondrial adaptations may have occurred quickly enough to have contributed to the increase in VO2maxafter 5 days(HIIT-D)or 2 weeks (HIIT-S) by increasing maximum arteriovenous oxygen difference.35Moreover,the training-induced improvements in VO2maxin such a short time are also associated with the augmented red blood cell volume,resulting in higher O2-carrying capacity and stroke volume and,hence,O2transport capacity.38Increases in skeletal muscle peroxisome-proliferator activated receptor γ coactivator-1α (PGC-1α),which is a major regulator of exercise-induced mitochondrial biogenesis and substrate utilization in the muscle,have been observed after 2 weeks of HIIT.10,31Indeed,increases in skeletal muscle mitochondrial biogenesis and mitochondrial enzyme activity (e.g.,citrate synthase activity) have been observed after 2 weeks of HIIT in normal healthy individuals,7－10,39unhealthy populations,31and even highly trained athletes.40Considering that a single session of HIIT activates adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase,41which phosphorylate and activate PGC-1α and turns on mitochondrial biogenesis,16,41it will be important to determine if there are increases in mitochondrial biogenesis and mitochondrial enzyme activities even after the second day of 5 days of HIIT-D.

In summary,5 days of HIIT training increases VO2max,and previous literature on studies involving 2 weeks of HIIT suggests that these increases occur without increases in maximum cardiac output.Further studies are required to determine the ways in which 5 days of HIIT increases VO2max.

4.2.HIIT and substrate oxidation during submaximal exercise

Previous studies have found that 2 or more weeks of HIIT results in more eff icient energy utilization during submaximal exercise.More eff icient energy use is indicated by less skeletal muscle glycogen use,11,17less muscle lactate production,11lower blood lactate levels,42lower RER,11and attenuated hormonal responses such as insulin,epinephrine,norepinephrine,and glucose.11,31The mechanism for this improved aerobic prof ile during submaximal exercise is likely to be the observed increase,after HIIT,in skeletal muscle oxidative enzymes,8,10,12,31increases in enzymes related to fat oxidation such as cluster of differentiation 36,carnitine palmitoyltransferase I,and beta-hydroxyacyl-CoA-dehydrogenase17,39and reduced lactate dehydrogenase activity,39which is likely due to the observed increase in PGC-1α and other regulatory factors.4,30Remarkably,we found that just 5 days of HIIT-D was as effective as 2 weeks of HIIT-S in increasing fat oxidation and decreasing CHO oxidation during submaximal exercise.No other study has shown such rapid benef icial effects on submaximal exercise metabolism of HIIT,and it will be important for follow-up studies to determine if skeletal muscle adaptations and/or hormonal changes are involved.It is possible that the lower skeletal muscle glycogen levels during the second bout of HIIT (only 3 h after the first HIIT bout) may be a stimulus that results in more rapid skeletal muscle adaptations.It is very unlikely that the meal provided to the HIIT-D group following the first HIIT session would have replaced a substantial amount of the glycogen.17

Although no sprint interval training or HIIT studies have examined whether skeletal muscle metabolic or hormonal changes occur after only 5－7 days of training,we are able to gain some insight from studies that involved endurance training.Indeed,it has been reported that 3 consecutive days of exercise eliciting～60% of VO2maxincreases skeletal muscle glucose and lactate transporters.13Phillips et al.14found that 5 days of cycling 2 h/day at 60%of VO2peakincreased total fat oxidation by 10% and decreased glycogen oxidation by 16%during 90 min of submaximal exercise.14

4.3.HIIT and endurance capacity

We found that TTE was increased to a similar extent by either 6 sessions of HIIT over 2 weeks (HIIT-S) or by 6 sessions of HIIT over 5 days(HIIT-D).These results are in line with what others have found for improvements in TTE9and time trial performance9,10,17,18after 2 weeks of 4－6 repeated 30 s “all-out” Wingate cycling exercise bouts.Also,studies using the same HIIT protocol as used in our study(10×60 s at VO2max,interspersed by 75 s rest)have reported improvements in endurance capacity measured by TTE or time trial performance after 6 sessions of HIIT conducted over 2 weeks.8,10Remarkably,we found such improvements in exercise capacity after 6 sessions of HIIT over only 5 days(HIIT-D).

Studies with different HIIT protocols applied over 2 weeks have shown that such short-term,training-induced changes in endurance capacity are highly associated with peripheral adaptations.8,39This notion has been supported by evidence that no change in maximum cardiac output occurred despite increases in VO2maxand endurance capacity after 6 sessions of HIIT carried out over 2 weeks.8It has also been recently reported that a single day of HIIT administered twice in 1 day along with aerobic exercise is a potent stimulus for skeletal muscle signaling responses associated with mitochondrial biogenesis.16We are not aware of any study that reported that previously demonstrated improvements in endurance capacity induced by 6 sessions of HIIT over 2 weeks can be observed when the 6 sessions are administered over 5 days.As mentioned earlier,it is possible that the higher cellular stress of the twice-a-day approach (HIIT-D) used in our study may induce faster mitochondrial biogenesis adaptations than those induced by the standard HIIT approach of 6 sessions over 2 weeks(HIIT-S).12,16Indeed,Andrade-Souza et al.16recently showed that acute twice-a-day training can amplify the increase in genes associated with mitochondrial biogenesis.Additional studies are needed to reveal the molecular mechanisms driving the improvements in skeletal muscle oxidative capacity in the HIIT-D protocol.Given that CHO oxidation was reduced during prolonged submaximal exercise in both the HIIT-D and HIIT-S groups,it is likely that,compared to pre-training,exercise TTE was increased at least in part due to less glycogen use and lower lactate production during exercise.43

Our study had some limitations.For example,given that the data were collected from 28 recreational healthy males,it is not clear if similar results would occur if trained athletes were recruited.Moreover,our study involved males only,and recruiting both male and female participants would enable a comparison of whether there are gender differences in responses to the HIIT-D model.Our study was a proof of concept and did not involve mechanistic examinations that could be carried out with muscle biopsies.Therefore,it will be important for follow-up studies to examine skeletal muscle molecular adaptations to determine if similar mechanisms are at play in HIIT-D and HIIT-S.

5.Conclusion

We demonstrated for the first time that 6 sessions of HIIT over only 5 days (HIIT-D) increased VO2max,endurance capacity,and submaximal exercise fat oxidation similar to those achieved by 6 sessions of HIIT over 2 weeks (HIIT-S).Our findings make this study the shortest HIIT study (5 days)that we are aware of.This demonstrates the remarkable adaptability of the human body to exercise stress.Therefore,coaches and athletes may wish to apply the HIIT-D model to improve endurance and substrate oxidation during exercise.

Acknowledgments

This study was funded by a grant from Hacettepe University Scientif ic Research Projects Coordination Unit (Project ID:TSA-2019-16811).MMA was supported with a PhD scholarship by the Scientif ic and Technological Research Council of Turkey(TUBITAK).The authors are grateful to all participants involved in the study and would like to thank Derya Canan Korur,Selin Aktitiz,Dilara Kuru,Nesli¸sah Tortop,and ˙Irem Güngör for their assistance with data collection.

Authors’contributions

MMA designed the study,collected the data,and wrote the manuscript;HHT was involved in developing the study idea,constructing the methods,reviewing the data analyses,and writing the manuscript;YG was involved in the data collection and constructing the methods;SB contributed to the study design and data collection;ŞNK contributed to the development of the study idea,data collection,and manuscript preparation;GKM critically reviewed the data and contributed to manuscript preparation.All authors have read and approved the final version of the manuscript,and agree with the order of presentation of the authors.

Competing interests

The authors declare that they have no competing interests.