Effects of Sprint Interval Training at Different Altitudes on Cycling Performance at Sea-Level

Background: Bene ts of sprint interval training performed in hypoxia (SIH) compared to normoxia (SIN) have been assessed by studies mostly conducted around 3000 m of simulated altitude. The present study aims to determine whether SIH at an altitude as high as 4000 m can elicit greater adaptations than the same training at 2000 m, 3000 m or sea-level. Methods: Thirty well-trained endurance male athletes (18–35 years old) participated in a six-week repeated sprint interval training program (30 s all-out sprint, 4 min 30 s recovery; 4–9 repetitions, 2 sessions/week) at sea-level (SL, n=8), 2000 m (FiO216.7%,n=8), 3000 m (FiO214.5%,n=7) or 4000 m (FiO213.0%,n=7). Aerobic and anaerobic exercise components were evaluated by an incremental exercise test, a 600 kJ time trial and a Wingate test before and after the training program. Results: After training, peak power output (PPO) during the incremental exercise test increased (~6%) without di erences between groups. The lactate threshold assessed by Dmax increased at 2000 m (+14 12 W) and 4000 m (+12 11 W) but did not change at SL and 3000 m. Mean power during the Wingate test increased at SL, 2000 m and 4000 m, although peak power increased only at 4000 m (+38 38 W). Conclusions: The present study indicates that SIH using 30 s sprints is as e cient as SIN for improving aerobic and anaerobic qualities. Additional bene ts such as lactate-related adaptations were found only in SIH and Wingate peak power only increased at 4000 m. This nding is of particular interest for disciplines requiring high power output, such as

38 W). Conclusions: The present study indicates that SIH using 30 s sprints is as e cient as SIN for improving aerobic and anaerobic qualities. Additional bene ts such as lactate-related adaptations were found only in SIH and Wingate peak power only increased at 4000 m. This nding is of particular interest for disciplines requiring high power output, such as in very explosive sports. Keywords:hypoxia; cycling; lactate threshold; repeated sprint training; time trial; Wingate test 1. Introduction Hypoxic training has been used to enhance cycling performance at sea-level for decades. Originally, altitude training camps were organized at moderate altitude (1800–2500 m) for two to four weeks, two or three times a year. This method has been de ned as the traditional “Live high–Train high”. Since then, various strategies have been proposed such as “Live high–Train low”, “Intermittent hypoxic exposure” (IHE) or “Intermittent hypoxic training” (IHT) [1]. This last method has gained in popularity especially when combined with high intensity interval training (HIIT). Even if the mechanisms by which hypoxic training has additive e ects compared to classical training at sea level remain unclear, some evidence indicates that both aerobic and anaerobic components could bene t from IHT [2,3]. In the early 2000s, a new variation of HIIT consisting of the repetition of short ( 30 s) all-out sprints emerged [4,5]. For a much smaller training volume, sprint training has demonstrated to be as e ective as continuous endurance training to increase muscle oxidative capacity [4,6–8], maximal Sports2020,8, 148; doi:10.3390 /sports8110148 /journal/sports

Sports2020,8, 148 2 of 13 oxygen uptake (VO2max) [4,7] and cycling time trial performance [6,8]. The two major sprint training protocols can be described as follows: repeated sprint training (RST) is characterized by repeated maximal exercise bouts of short duration ( 10 s) interspersed with brief recovery periods ( 60 s or exercise-to-rest ratio<1:4), whereas sprint interval training (SIT) includes longer sprints (usually 30 s) with 2–4 min recovery [9,10]. When performed in hypoxia, these types of training are de ned as RST in hypoxia (RSH) or SIT in hypoxia (SIH). The current study will focus on the latter. Although single aerobic and anaerobic performance seem to be improved in a similar way with SIH in comparison to the same training performed in normoxia (SIN), some speci cities have been highlighted [11]. SIH enhanced cycling power output at 4 mmol L 1 lactate during an incremental exercise test [12] as well as the ventilatory threshold [13] when no improvement was reported with SIN. Although altitude training camps and IHE seem to be e cient for enhancing hematocrit, hemoglobin mass and red blood cells, most of the studies investigating IHT do not report any hematological changes [1]. Being involved in numerous physiological processes such as oxidative metabolism or erythropoiesis, iron is essential for endurance athletes whose athletic performance requires a high aerobic capacity [14,15]. The decline of iron storage in the blood is well documented as a result of altitude training camp [15,16], however there is a lack of data regarding the e ects of sprint training protocols on ferritin levels. Short all-out sprint (<45 s) performance does not appear to be impaired by acute hypoxia. Nevertheless, the decrease in oxygen availability is compensated by an increase in the production of ATP by glycolysis [17]. Due to the lower rate of oxygen delivery, it has been hypothesized that SIH induces a higher expression and activity of glycolytic enzymes, which in turn could enhance performance during a Wingate test or a time trial. In line with the latter, six weeks SIH, but not SIT, increased phosphofructokinase (PFK) activity, despite similar improvement in

ATP by glycolysis [17]. Due to the lower rate of oxygen delivery, it has been hypothesized that SIH induces a higher expression and activity of glycolytic enzymes, which in turn could enhance performance during a Wingate test or a time trial. In line with the latter, six weeks SIH, but not SIT, increased phosphofructokinase (PFK) activity, despite similar improvement in cycling performance in both groups [12]. The authors hypothesized that other adaptations, such as bu er capacity, might be required to detect an additional e ect of SIH over SIT on cycling performance [12]. To date, most of the SIH studies have been conducted around 3000 m of simulated altitude [12,13,18,19]. To our knowledge, no study has shown that this altitude is optimal for maximizing the performance improvements induced by SIH programs. The present study aims to determine whether SIH at an altitude as high as 4000 m can elicit a greater physiological stress and therefore larger adaptations than the same training at 2000 m, 3000 m or sea-level. 2. Materials and Methods 2.1. Participants A sample size analysis has been performed according to the superiority formula for parallel RCT with continuous variables and the calculator developed by Wang and Ji [20]. To nd a 10% di erence in the means of peak power output (PPO) between pre- and post-training with an SD corresponding to 7% [18] and a 2% superiority margin, as well as power of 80% and a signi cance level of 5%, considering 3 treated groups were tested and compared to 1 control group, it was predicted that 28 participants (n=7/group) were needed if a drop-out rate of 3% was taken into account. The superiority margin represents the threshold above which a group was considered as gaining an advantage from one altitude over another. Thirty-one well-trained endurance male athletes (cyclists or triathletes) competing in amateur categories gave their written consent to voluntarily participate in the experiment, which was approved by the ethical committee of the UCLouvain (B403201939034) and conducted in accordance with the Declaration of Helsinski. The participants were recruited based on their age (18–35 years) and

an advantage from one altitude over another. Thirty-one well-trained endurance male athletes (cyclists or triathletes) competing in amateur categories gave their written consent to voluntarily participate in the experiment, which was approved by the ethical committee of the UCLouvain (B403201939034) and conducted in accordance with the Declaration of Helsinski. The participants were recruited based on their age (18–35 years) and their training volume (4 to 10 h/week). Exclusion criteria for participation were smoking, exposure to an altitude above 1500 m during the month before the experiment, and any health risk that could compromise the participant's safety during training and/or hypoxic exposure. The participants were asked to maintain their usual dietary habits and training load throughout the experiment. They completed a medical survey to ensure they were not taking any medication or

Sports2020,8, 148 3 of 13 supplements that could interfere with the experiment. Only 2 participants reported occasional whey protein consumption, and none took iron supplementation. They were also asked to avoid strenuous workouts the day before the experimental training sessions. The participants recorded all their personal workouts in a Strava diary to allow us to quantify their training volume. Of the thirty-one participants involved, only one had to stop the study—due to a fall that occurred during a personal training session. 2.2. Experimental Protocol The study was conducted between September 2019 and January 2020 in 2 experimental periods. During each period, 3 phases were organized: 2 weeks of pre-testing, 6 weeks of training and 2 weeks of post-testing. All pre- and post-tests were performed at sea-level. All cycling tests and training sessions were performed using a cycle ergometer (Cyclus II; RBM Electronics, Leipzig, Germany) and the participant's personal bike. The pre-testing period consisted of 5 visits to the laboratory, which can brie y be summarized as follows. First visit (day 1): height, weight and hemoglobin mass measurement, blood sampling. Second visit (day 3): incremental test and familiarization with the Wingate test. Third visit (day 4): familiarization with the 600 kJ time trial. Fourth visit (day 8): Wingate test. Fifth visit (day 10): 600 kJ time trial. After the pre-test period, the participants were semi-randomly assigned to one of the 4 experimental groups based on the PPO measured during the incremental exercise test to obtain similar values of tness level, age, and body weight (Table). The 4 groups participated in a 6-week supervised cycling training program involving SIT at di erent altitudes: sea-level (SL, FiO220.9%,n=8), 2000 m (FiO216.7%,n=8), 3000 m (FiO214.5%,n=7) and 4000 m (FiO213.0%,n=7). Table 1.Participant characteristics. Sea-Level (n=8) 2000 m (n=8) 3000 m (n=7) 4000 m (n=7) Age (year) 26.0 4.4 25.4 4.6 25.4 4.2 25.1 4.5 Weight (kg) 67.0 4.1 71.2 7.6 72.4 9.9 72.6 12.4 Basal PPO (W) 313 31 318 29 335 31 307 29 Values are means SD for sea-level group (FiO220.9%,) and altitude groups (2000 m, FiO216.7%; 3000 m, FiO214.5%; 4000 m, FiO213.0%). Hypoxia

m (n=8) 3000 m (n=7) 4000 m (n=7) Age (year) 26.0 4.4 25.4 4.6 25.4 4.2 25.1 4.5 Weight (kg) 67.0 4.1 71.2 7.6 72.4 9.9 72.6 12.4 Basal PPO (W) 313 31 318 29 335 31 307 29 Values are means SD for sea-level group (FiO220.9%,) and altitude groups (2000 m, FiO216.7%; 3000 m, FiO214.5%; 4000 m, FiO213.0%). Hypoxia was achieved following nitrogen injection into a con ned room to dilute oxygen content in the air (High altitude system B-Cat, Tiel, The Netherlands). After the training intervention, all participants participated to the post-test period, which was identical to the pre-test without the familiarization sessions. Blood sampling was performed 3 days after the last training session and the rst cycling test was performed 5 days after the last session to allow a complete recovery. 2.3. Training Sessions All participants were blind to the conditions until the end of the experiment. They participated in a supervised SIT session twice a week for 6 weeks based on methods from Puype et al. [12]. The sessions started with a 10 min warm-up at ~150 W (80–90 revolutions per minute (RPM), 17.5 Nm) and two 5 s sprints. Thereafter, the participants had to repeat a series of 30 s sprints with a 4 min 30 s-recovery at ~75 W (80–90 RPM, 8.75 Nm). The rst and last sprint of each session were set as a Wingate test (see below). The other sprints were set at 80% of the Wingate load. The participants were asked to `go all-out' on all sprints and try to produce the highest possible power output. The number of sprints per session was progressively increased from 4 in week 1 to 9 in week 6. 2.4. Blood Analyses Hematocrit (Hct) and hemoglobin concentrations ([Hb]) were measured immediately after blood drawing from an antecubital vein of the forearm using an automated device (ABX Micros 60, Axonlab, Dättwil, Switzerland). EDTA tubes were then centrifuged for 15 min at 2000 gat 4 C and plasma

hemoglobin concentrations ([Hb]) were measured immediately after blood drawing from an antecubital vein of the forearm using an automated device (ABX Micros 60, Axonlab, Dättwil, Switzerland). EDTA tubes were then centrifuged for 15 min at 2000 gat 4 C and plasma

Sports2020,8, 148 4 of 13 fraction was collected and stored at 20 C. Plasma ferritin concentrations were also measured using an automated analyzer (Pentra C 200, Horiba medical, Axonlab, Dättwil, Switzerland). Blood, plasma and red blood cell volumes (BV, PV and RBCV) were calculated based on hematocrit, hemoglobin concentrations and hemoglobin mass (see section below) [21,22]: RBCV=Hbmass/MCHC 100 (1) BV=RBCV (100/Hct) (2) PV=BV – RBCV (3) where MCHC is the mean corpuscular hemoglobin concentration (([Hb]/Hct) 100), and Hbmassis the hemoglobin mass. 2.5. Hemoglobin Mass Measurement Hemoglobin mass was measured using a modi ed version of the optimized carbon monoxide (CO) rebreathing method developed by Schmidt and Prommer [23] and detailed in Meurrens et al. [24]. The reliability of the method is characterized by a typical error of ~1.4% [22,23,25] and the validity of the procedure has been con rmed after a close agreement between the measured and calculated losses of total Hbmassvia 550 mL phlebotomy (mean error of 9 g) was found [23]. 2.6. Incremental Exercise Test A maximal incremental exercise test was performed to assess aerobic tness, as determined by VO2maxand PPO. The starting load was set at 110 W and was incremented by 40 W every 3 min until exhaustion. Heart rate (HR) (Polar Team System 2; Polar Electro, Kempele, Finland) and respiratory exchanges (Ergocard Clinical, Medisoft, Sorinnes, Belgium) were continuously monitored while a capillary blood sample was collected from the right earlobe in the last 15 s of each stage for the determination of blood lactate concentrations (Lactate Pro, Arkray, Japan). Based on the lactate curve, the power produced at Dmax was calculated according to Cheng et al. (1992) [26]. Due to technical issues, VO2maxresults cannot be presented. 2.7. Wingate Test The Wingate test consisted of a 30 s maximal cycling exercise with a resistance set to 7.5% of the participant's body weight (0.075 kg kg bw 1 ). The test was preceded by a 10 min warm-up without resistance including two 5 s sprints at a test load. The participants were asked to try and generate the highest power possible throughout the test and were strongly

30 s maximal cycling exercise with a resistance set to 7.5% of the participant's body weight (0.075 kg kg bw 1 ). The test was preceded by a 10 min warm-up without resistance including two 5 s sprints at a test load. The participants were asked to try and generate the highest power possible throughout the test and were strongly encouraged during the 30 s. The following variables were subsequently calculated: peak power, mean power for the 30 s, and the fatigue index representing the decrease in power during the test (W s 1 ). Blood lactate concentrations were measured 3 min and 5 min post-Wingate (Lactate Pro, Arkray, Japan). 2.8. 600 kJ Time Trial (TT) To simulate a eld performance, the participants performed a 600 kJ TT. After a 10 min warm-up at 100 W, the participants had to reach 600 kJ as quickly as possible. The only information given to the participants was the work progression in kJ and the RPM. 2.9. Statistics All values are expressed as the means SD. Repeated-measures ANOVA were performed on raw values considering the condition (SL, 2000 m, 3000 m or 4000 m) as the inter-group factor, and time (pre-training and post-training) as an intra-group factor. Tukey post hoc analyses were performed when indicated. Pre- and post-training measurements for each group were compared using a two-tailed paired Student'st-test with 95% con dence interval (CI). All analyses resulting inp<0.05 were

Sports2020,8, 148 5 of 13 considered to be statistically signi cant, whilep-values between 0.05 and 0.10 were described as tendencies. The above statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS v.25.0; IBM, Armonk, NY, USA). E ect size (ES) was determined using Cohen's d developed by Cohen (1988) [27] and discussed in Lakens (2013) [28]. The magnitude of the ES was classi ed as huge (>2.0), very large (1.19–2.0), large (0.80–1.19), medium (0.50–0.79), small (0.20–0.49) or very small (<0.19) [29]. 3. Results 3.1. Incremental Exercise Test PPO increased after training without di erences between groups (SL:+20 18 W, t=3.09, CI=[4.58;34.42],p=0.018, d=0.95; 2000 m:+22 19 W, t=3.38, CI=[6.63;37.62],p=0.012,d=0.92; 3000 m:+16 13 W, t=3.4, CI=[4.55;28.02],p=0.015, d=0.94; 4000 m:+20 11 W, t=4.72, CI=[9.7;30.59],p=0.003, d=0.91) (FigureA). Regarding lactate, a main training e ect for power at Dmax was found (p=0.001, FigureB). Post-hoc analyses revealed that power at Dmax increased only at 2000 m (+14 12 W, t=3.29, CI=[3.91;23.9],p=0.013, d=0.94) and 4000 m (+12 11 W, t=2.74,CI=[1.28;22.54],p=0.034, d=0.93) (FigureB). The maximal blood lactate concentrations increased after training (main e ect,p=0.05) and the interaction between training and groups tended to be signi cant (main e ect,p=0.063) (FigureC). Post-hoc analyses revealed that maximal lactate concentrations increased after training at SL (+2.5 2.4 mmol L 1 , t=3.02, CI=[0.55;4.5],p=0.019, d=0.68) and tended to increase at 2000 m (+2.4 3.6 mmol L 1 , t=1.9, CI=[ 0.6;5.45],p=0.1, d=0.67). Finally, no changes in maximal heart rate were detected in any conditions (FigureD).Sports 2020, 8, x FOR PEER REVIEW 5 of 13 Cohen’s d developed by Cohen (1988) [27] and discussed in Lakens (2013) [28]. The magnitude of the ES was classified as huge (>2.0), very large (1.19–2.0), large (0.80–1.19), medium (0.50–0.79), small (0.20–0.49) or very small (<0.19) [29]. 3. Results 3.1. Incremental Exercise Test PPO increased after training without differences between groups (SL: +20 ± 18 W, t = 3.09, CI = [4.58;34.42], p = 0.018, d = 0.95; 2000 m: +22 ± 19 W, t = 3.38, CI = [6.63;37.62], p = 0.012, d = 0.92; 3000 m: +16 ± 13 W, t =

small (<0.19) [29]. 3. Results 3.1. Incremental Exercise Test PPO increased after training without differences between groups (SL: +20 ± 18 W, t = 3.09, CI = [4.58;34.42], p = 0.018, d = 0.95; 2000 m: +22 ± 19 W, t = 3.38, CI = [6.63;37.62], p = 0.012, d = 0.92; 3000 m: +16 ± 13 W, t = 3.4, CI = [4.55;28.02], p = 0.015, d = 0.94; 4000 m: +20 ± 11 W, t = 4.72, CI = [9.7;30.59], p = 0.003, d = 0.91) (Figure 1A). Regarding lactate, a main training effect for power at Dmax was found (p = 0.001, Figure 1B). Post-hoc analyses revealed that power at Dmax increased only at 2000 m (+14 ± 12 W, t = 3.29, CI = [3.91;23.9], p = 0.013, d = 0.94) and 4000 m (+12 ± 11 W, t = 2.74, CI = [1.28;22.54], p = 0.034, d = 0.93) (Figure 1B). The maximal blood lactate concentrations increased after training (main effect, p = 0.05) and the interaction between training and groups tended to be significant (main effect, p = 0.063) (Figure 1C). Post-hoc analyses revealed that maximal lactate concentrations increased after training at SL (+2.5 ± 2.4 mmol·L −1 , t = 3.02, CI = [0.55;4.5], p = 0.019, d = 0.68) and tended to increase at 2000 m (+2.4 ± 3.6 mmol·L −1 , t = 1.9, CI = [−0.6;5.45], p = 0.1, d = 0.67). Finally, no changes in maximal heart rate were detected in any conditions (Figure 1D). Figure 1. Incremental exercise test. (A) Peak power output (PPO); (B) Power at Dmax; (C) Maximal lactate concentrations; (D) Maximal heat rate (HR max). Values are means ± SD pre- and post-training. * p < 0.05; ** p < 0.01 vs pre-training. Figure 1. Incremental exercise test. (A) Peak power output (PPO); (B) Power at Dmax; (C) Maximal lactate concentrations; (D) Maximal heat rate (HRmax). Values are means SD pre- and post-training. *p<0.05; **p<0.01 vs pre-training.

This research investigates the effects of sprint interval training at various altitudes on cycling performance.