Effects of Intermittent Hypoxic Training on Aerobic Capacity and Second Ventilatory Threshold in Untrained Men

he aim of study was to evaluate the effects of interval training performed in hypoxia on aerobic capacity and second ventilatory threshold in young, untrained men. Participants (n = 48) were randomly divided into a control group and two groups performing the same interval training (three times a week for 4 weeks) in normoxia (200 m asl) (NT) and in hypoxia (IHT) (3000 m asl, FIO 2= 14.4% ). In the incremental test, maximal oxygen uptake (VO 2max) was measured and the rst (VT1) and second (VT2) ventilatory thresholds and the maximal power output (Pmax) were determined for each participant. The training workloads of the efforts corresponded to the workload at VT2 (effort) and VT1 (active recovery). Training in both normoxia and hypoxia signi cantly increased absolute VO 2max(p= 0.02,

the incremental test, maximal oxygen uptake (VO 2max) was measured and the rst (VT1) and second (VT2) ventilatory thresholds and the maximal power output (Pmax) were determined for each participant. The training workloads of the efforts corresponded to the workload at VT2 (effort) and VT1 (active recovery). Training in both normoxia and hypoxia signi cantly increased absolute VO 2max(p= 0.02, ES = 0.51 andp= 0.002, ES = 0.47, respectively). In comparison to NT, only IHT signi cantly (p< 0.001; ES = 0.80) improved Pmax, as well as power at VT2 (p= 0.02; ES = 0.78). The applied IHT was effective in improving Pmax and power at VT2, which was not observed after training in normoxia. Keywords: hypoxia; interval training; maximal oxygen uptake; anaerobic threshold; training; performance 1. Introduction Physiological determinants of performance in endurance sports are maximal oxygen uptake (VO2max), oxygen consumption at lactate thresholds (ventilatory thresholds), velocity (or power output) at metabolic thresholds, submaximal economy and percentage of slow-twitch muscle bers [1,2]. As exercise intensity increases up to maximal (VO2max), exercise metabolism changes. When the intensity exceeds the rst ventilatory threshold (VT1), metabolic acidosis develops and this threshold appears to be a sensitive indicator of the development of metabolic acidosis [3], and at VT1, there is the greatest intensity of aerobic metabolism. The second ventilatory threshold (VT2) (respiratory compensation point) is useful in the evaluation of aerobic performance [4,5] because exceeding intensity at VT2 induces lactate accumulation in the blood and hyperventilation, leading to fatigue caused by decompensated metabolic acidosis [6]. In sports, special attention is given to VO2max and VT2 regarding training methods, supplementation or environmental conditions that increase VO2max and oxygen uptake and power (or speed) at VT2. Recently, one of the most popular methods of supporting physical training is the inclusion of hypoxia in training [7,8]. Physiological adaptations in response to hypoxia are well documented and include increases in maximal aerobic capacity, capillary density, hemoglobin mass and erythrocyte count due to increased erythropoietin Appl. Sci.2023,13, 9954.

physical training is the inclusion of hypoxia in training [7,8]. Physiological adaptations in response to hypoxia are well documented and include increases in maximal aerobic capacity, capillary density, hemoglobin mass and erythrocyte count due to increased erythropoietin Appl. Sci.2023,13, 9954.

Appl. Sci.2023,13, 9954 2 of 10 production under hypoxia. A number of hypoxic training protocols have been developed with varying effectiveness by using different combinations of training at varying altitudes and utilizing both natural and arti cial altitudes [9]. Training under hypobaric hypoxia is often difficult to implement due to travel time, cost and athlete engagement [10]. For this reason, the live low–train high (LL-TH) training method is gaining popularity and becoming an important method of hypoxic training in modern sports. Intermittent hypoxic training (IHT) is a method in which athletes live at low altitudes, but train under hypoxic conditions (LL-TH), and it is believed that IHT can enhance performance improvements compared to similar training at sea level [11]. In this method, the athlete stays in hypoxia only for training but spends most of the time during the day in normoxia. IHT causes only minor disruptions to athletes' daily routines, allowing them to maintain their normal lifestyles [12]. Importantly, the quality of sleep and recovery is well preserved, as athletes do not sleep in hypoxic conditions [10]. However, published data reported inconclusive effectiveness of IHT in improving VO2max and hematological parameters [13,14]. After IHT, improvements in aerobic and anaerobic capacity are reported [15,16], no effects [17] or even negative effects of this training are indicated (reduced speeds and power output, reduced oxygen flux in athletes) [18]. Treff et al. [19] recently emphasized that, despite the extensive literature, clear evidence and practical guidance on how hypoxic training allows to increase endurance at sea level is still lacking. The two main methods of endurance training are moderate-intensity continuous train- ing and interval training. Interval exercise typically involves repeated bouts of relatively intense exercise interspersed by short periods of recovery. These two methods have dif- ferent ef ciencies in improving endurance performance and still not shown what is the optimal model of interval training in terms of intensity, duration and frequency to induce improvements in endurance [20]. The choice of training workload in interval training is usually made in relation to VO2max, maximal heart rate (HRmax) or maximal work- load [20], which does

have dif- ferent ef ciencies in improving endurance performance and still not shown what is the optimal model of interval training in terms of intensity, duration and frequency to induce improvements in endurance [20]. The choice of training workload in interval training is usually made in relation to VO2max, maximal heart rate (HRmax) or maximal work- load [20], which does not take into account the exercise metabolism (metabolic thresholds). In this study, to improve endurance performance, we proposed a novel interval training method based on power at VT1 and VT2, which is additionally supported by normobaric hypoxia (IHT). We hypothesized that such a training model would be more effective in the improvement of endurance performance than in interval training performed in normoxia. The purpose of this study was to determine the effectiveness of interval training performed in hypoxia on maximal oxygen uptake and second ventilatory threshold. 2. Methods Only healthy men (without medical contraindications to physical exercise) were re- cruited for the study. To con rm the absence of contraindications to physical training, before intervention, participants underwent a medical quali cation procedure (e.g., elec- trocardiogram at rest and during exercise, complete blood count, hemoglobin and iron concentration). Exercise tests were performed under the supervision of a sports medicine physician. Participants were not allowed to participate in regular physical training for 6 months before the intervention. The sample size was estimated using G*Power 3.1 (Dusseldorf, Germany) and the required sample size was 16 participants per group. Participants (n = 48) were randomly divided into three groups: the control group (ctrl) and 2 groups performing the same interval training (3 times a week for 4 weeks): in normoxia (NT) (200 m asl) and in hypoxia (IHT) (3000 m asl, FIO2= 14.4%). Before and 7–10 days after the training, the participants performed the exercise incremental test to volitional exhaustion. The incremental test was conducted in normoxia. Prior to the intervention, participants were familiarized with the laboratory, testing equipment and procedures, as well as the purpose of the study. They were also instructed on how to prepare for the graded test and training, i.e.,

and 7–10 days after the training, the participants performed the exercise incremental test to volitional exhaustion. The incremental test was conducted in normoxia. Prior to the intervention, participants were familiarized with the laboratory, testing equipment and procedures, as well as the purpose of the study. They were also instructed on how to prepare for the graded test and training, i.e., avoid heavy physical exertion and dehydration. They should also sleep for at least 6–8 h the night before the test/training and eat a light meal at least 2 h before the test/training. Participants were recommended not to

Appl. Sci.2023,13, 9954 3 of 10 change their habitual diet and not to engage in any additional physical activity during the intervention. Participation in the study was voluntary, and participants at any stage of the project were allowed to opt out of further participation. All study participants signed a consent form to participate in the project. The project was approved by the Bioethical Commission of the Regional Medical Chamber in Krakow, Poland (opinion No. 47/KB/OIL/2022). All test procedures were conducted in accordance with the principles adopted in the Declaration of Helsinki. 2.1. Participants The study involved healthy, moderately physically active men, but not ones who regularly practiced any sport. Their physical activity was varied and spontaneous. The participants were between 19 and 26 years old and the groups did not differ in body composition. Body mass and body composition did not change signi cantly after training compared to baseline (Table). Table 1. Age and anthropometric measurements of the participants (data are presented as mean SD). Variable Group Pre Post Effect: Group F p p 2 Effect: Time F p p 2 Interaction F p p 2 Age (yrs) ctrl 22.9 2.9 - - - -NT 20.5 1.0 - IHT 21.5 1.5 - BH (cm) ctrl 178.9 5.9 - - - -NT 179.7 5.6 - IHT 182.0 5.5 - BM (kg) ctrl 75.8 11.5 75.9 11.7 0.55 0.58 0.02 0.07 0.80 0.01 0.71 0.49 0.03 NT 76.7 8.3 76.9 8.2 IHT 79.6 10.4 79.2 9.6 LBM (kg) ctrl 61.5 7.3 61.9 7.7 1.45 0.24 0.05 2.49 0.12 0.05 0.55 0.57 0.02 NT 63.1 6.0 63.1 6.1 IHT 65.6 7.5 66.1 7.9 FAT (%) ctrl 18.3 5.3 18.0 5.0 0.35 0.70 0.01 3.27 0.08 0.06 2.25 0.11 0.09 NT 17.6 3.9 17.7 4.4 IHT 17.4 3.9 16.3 3.8 FM (kg) ctrl 14.3 5.4 14.0 5.2 0.07 0.92 0.003 3.20 0.08 0.06 2.44 0.1 0.09 NT 13.7 3.9 13.8 4.4 IHT 14.1 4.5 13.0 4.1 BMI ctrl 23.6 2.9 23.7 2.9 0.04 0.95 0.001 0.12 0.72 0.002 0.55 0.57 0.02 NT 23.7 2.2 23.8 2.2 IHT 24.0 2.3 23.9 2.1 BH: body height;

4.4 IHT 17.4 3.9 16.3 3.8 FM (kg) ctrl 14.3 5.4 14.0 5.2 0.07 0.92 0.003 3.20 0.08 0.06 2.44 0.1 0.09 NT 13.7 3.9 13.8 4.4 IHT 14.1 4.5 13.0 4.1 BMI ctrl 23.6 2.9 23.7 2.9 0.04 0.95 0.001 0.12 0.72 0.002 0.55 0.57 0.02 NT 23.7 2.2 23.8 2.2 IHT 24.0 2.3 23.9 2.1 BH: body height; BM: body mass; LBM: lean body mass; FM: fat mass; BMI: body mass index; ctrl: control group; NT: normoxia training; IHT: intermittent hypoxia training. 2.2. Somatic Measurements Prior to the rst and second graded test, participants underwent somatic measure- ments including body height, body mass (BM) and body composition. Fat percentage (%FAT), fat mass (FM), lean body mass (LBM) and body mass index (BMI) were deter-

Appl. Sci.2023,13, 9954 4 of 10 mined. Body height was measured with a stadiometer (Seca 217, Hamburg, Germany), whereas body mass and body composition were measured with a body composition ana- lyzer using the bioelectrical impedance method (Jawon Medical, IOI-353, Seoul, Republic of Korea). 2.3. Physical Activity and Diet A seven-day physical activity recall (PAR) questionnaire was used to assess partici- pants' physical activity [21,22]. Participants were instructed on the purpose of the study and how to complete the questionnaire. The interview was conducted by the researcher, who clari ed all the participants' doubts about the answers to the questions in real time. The data presented physical activity before the intervention. The caloric intake of the diet was estimated using the Fitatu (version 3.37, Poznan, Poland) application [23]. After familiarizing with application, participants kept dietary diaries for 7 days, entering all foods consumed into the application. Caloric consumption is presented as caloric intake per week (kcal/week). 2.4. Aerobic Capacity and Ventilatory Thresholds Aerobic capacity was examined by direct method (cardiopulmonary exercise test) using a ramp protocol in a graded test. Participants performed the test on an eBike Comfort bicycle ergometer (GE Health Care, Chicago, IL, USA) until volitional exhaustion. The test began with a 4-min warm-up at 60 watts and then power was systematically increased by 15 watts/min. In the incremental test, oxygen uptake (VO2), heart rate (HR), respiratory exchange ratio (RER), pulmonary ventilation (VE), carbon dioxide output (VCO2), fractional concentrations of expired CO2(%FECO2) and O2(%FEO2), ventilatory equivalent ratio for oxygen (VE/VO2) and carbon dioxide (VE/VCO2) were measured breath by breath using an ergospirometer (MetaLyzer 3R, Cortex, Germany). The rst and second ventilatory threshold, maximal power output (Pmax), power output at ventilatory thresholds, maximal oxygen uptake and maximal heart rate were determined for each participant. The results were analyzed by an experienced exercise physiologist who evaluated the changes of measured parameters with increasing work load. The ventilatory thresholds were determined using the method of respiratory equivalents [24,25]. The rst threshold was detected by the power output at which the VE/VO2ratio and FEO2reached a minimum (nadir or rst increase of VE/VO2versus

were determined for each participant. The results were analyzed by an experienced exercise physiologist who evaluated the changes of measured parameters with increasing work load. The ventilatory thresholds were determined using the method of respiratory equivalents [24,25]. The rst threshold was detected by the power output at which the VE/VO2ratio and FEO2reached a minimum (nadir or rst increase of VE/VO2versus work load without a simultaneous increase in VE/VCO2versus work load). The second ventilatory threshold was detected by the power output at which the VE/VCO2ratio reached a minimum and the FECO2reached a maximum (nadir or nonlinear increase of VE/VCO2versus work load). To determine VO2max, the following criteria were applied: a plateau in VO2, RER > 1.1 and HR within 10 bpm of the age-predicted maximum. If no plateau was observed, but the rest of the criteria were met, VO2peak was taken as the VO2max [26]. The ergospirometer was calibrated according to the manufacturer's requirements (gas and volume calibration). 2.5. Training Interval training was performed in a hypoxic, thermoclimatic chamber (Hypoxico, Germany), or in normoxia, on bicycle ergometers (Wattbike, UK) and lasted 60 min. The training consisted of a warm-up (6 min) with power output at VT1 followed by 6 series of efforts lasting 6 min with active recovery of 3 min (2:1 ratio). The work load of the efforts was set individually and corresponded to the work load at VT2 (effort) and VT1 (active recovery). All training sessions took place at the same temperature of 21 0.5 C and humidity of 40 1%. 2.6. Statistical Analysis Data are presented as mean and standard deviation. Data distribution was checked using the Shapiro–Wilk test. Homogeneity of variance within the groups was tested via

Appl. Sci.2023,13, 9954 5 of 10 Levene's test. ANOVA with repeated measures or one-way ANOVA was used to assess the effects of training, effect size (partial eta-squared ( p 2 )) and inter-group difference. If a signi cant (p< 0.05) effect of the main factor (group, time or interaction group-time) was found in the ANOVA, post-hoc analysis was performed using the Tukey test. In post hoc analysis (if signi cant), the effect size (ES: Cohen's d) between baseline and post training was calculated. The effect size was interpreted as small (0.20), medium (0.50) or large (0.80) [ES], or as small (0.01), medium (0.06) or large (0.14) [ p 2 ] [27]. The STATISTICA 13 package (StatSoft, Inc., Tulsa, OK, USA) was used for calculations. 3. Results 3.1. Physical Activity and Diet The weekly caloric intake was similar (f = 0.16,p= 0.85, p 2 = 0.01) in all groups: NT: 21,233 3352 kcal/week, IHT: 20,707 3656 kcal/week and 21,291 2434 kcal/week in the control group. Physical activity also did not differ significantly for the groups (f = 0.43,p= 0.65, p 2 = 0.02). The average weekly energy expenditure in each group was: 21,108 2348 kcal/week (NT), 21,970 3062 kcal/week (IHT) and21,229 3014 kcal/week (ctrl). 3.2. Aerobic Capacity and Ventilatory Thresholds Training in both normoxia and hypoxia signi cantly increased absolute VO2max (p= 0.02, ES = 0.51 andp= 0.002, ES = 0.47, respectively; Figure). Relative oxygen uptake increased (p= 0.008) after training only in the IHT group (Table). In comparison to NT, only IHT signi cantly (p< 0.001; ES = 0.80) improved maximal power output (Table, Figure), as well as power output at VT2 (p= 0.02; ES = 0.78) (Table). Moreover, IHT also increased oxygen uptake at VT2, which was not observed after training in normoxia (Table).Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 of 11 RER ctrl 1.08 ± 0.06 1.12 ± 0.08 1.42 0.25 0.06 2.14 0.15 0.04 4.97 0.01 0.18 0.30 - NT 1.12 ± 0.04 1.09 ± 0.07 0.52 - IHT 1.11 ± 0.06 1.15 ± 0.08 0.25 - VO2max (L/min) ctrl 3.34 ± 0.43 3.38 ±

after training in normoxia (Table).Appl. Sci. 2023, 13, x FOR PEER REVIEW 6 of 11 RER ctrl 1.08 ± 0.06 1.12 ± 0.08 1.42 0.25 0.06 2.14 0.15 0.04 4.97 0.01 0.18 0.30 - NT 1.12 ± 0.04 1.09 ± 0.07 0.52 - IHT 1.11 ± 0.06 1.15 ± 0.08 0.25 - VO2max (L/min) ctrl 3.34 ± 0.43 3.38 ± 0.43 2.96 0.06 0.11 21.90 <0.001 0.32 3.05 0.05 0.11 0.96 0.09 NT 3.55 ± 0.37 3.74 ± 0.38 0.02 0.51 IHT 3.59 ± 0.50 3.85 ± 0.60 0.002 0.47 VO2max (mL/kg/min) ctrl 44.6 ± 6.0 45.1 ± 5.6 1.97 0.31 0.05 16.67 0.26 <0.001 2.57 0.08 0.10 0.99 0.08 NT 46.7 ± 5.8 49.1 ± 6.1 0.07 0.40 IHT 45.5 ± 7.2 48.7 ± 6.1 0.008 0.48 Pmax: maximal power output; HRmax: maximal heart rate; RER: respiratory exchange ratio; VO 2max: maximal oxygen uptake; ctrl: control group; NT: normoxia training; IHT: intermittent hy- poxia training. IHT control normoxia baseline post measurement 3,0 3,1 3,2 3,3 3,4 3,5 3,6 3,7 3,8 3,9 4,0 4,1 4,2 VO 2 max (L/min) * NS * Figure 1. Effect of interval training in normoxia (NT) and in hypoxia (IHT) on absolute maximal oxygen uptake (VO 2max) (*: p < 0.05; NS: non-significant; vertical bars indicate 0.95 confidence in- terval). Figure 1. Effect of interval training in normoxia (NT) and in hypoxia (IHT) on absolute maximal oxygen uptake (VO 2max) (*:p< 0.05; NS: non-significant; vertical bars indicate 0.95 confidence interval).

This study evaluates the impact of intermittent hypoxic training on aerobic capacity in untrained men.