Aerobic Continuous and Interval Training under Hypoxia Enhances Endurance Exercise Performance with Hemodynamic and Autonomic Nervous System Function in Amateur Male Swimmers

ypoxic training is often performed by competitive swimmers to enhance their perfor- mance in normoxia. However, the bene cial effects of aerobic continuous and interval training under hypoxia on hemodynamic function, autonomic nervous system (ANS) function, and en- durance exercise performance remain controversial. Here we investigated whether six weeks of aerobic continuous and interval training under hypoxia can improve hematological parameters, hemodynamic function, ANS function, and endurance exercise performance versus normoxia in amateur male swimmers. Twenty amateur male swimmers were equally assigned to the hy- poxic training group or normoxic training group and evaluated before and after six weeks of training. Aerobic continuous and interval training in the hypoxia showed a more signi cantly improved hemodynamic function (heart rate,

hematological parameters, hemodynamic function, ANS function, and endurance exercise performance versus normoxia in amateur male swimmers. Twenty amateur male swimmers were equally assigned to the hy- poxic training group or normoxic training group and evaluated before and after six weeks of training. Aerobic continuous and interval training in the hypoxia showed a more signi cantly improved hemodynamic function (heart rate, 653.4 vs. 353.7 beats/30 min; oxygen uptake, 62.45 vs. 16.22 mL/kg/30 min; stroke volume index, 197.66 vs. 52.32 mL/30 min) during sub- maximal exercise, ANS function (root mean square of successive differences, 10.15 vs. 3.32 ms; total power, 0.72 vs. 0.20 ms 2 ; low-frequency/high-frequency ratio, 0.173 vs. 0.054), and endurance exercise performance (maximal oxygen uptake, 5.57 vs. 2.26 mL/kg/min; 400-m time trial record, 20.41 vs. 7.91 s) than in the normoxia. These indicate that hypoxic training composed of aerobic continuous and interval exercise improves the endurance exercise performance of amateur male swimmers with better hemodynamic function and ANS function. Keywords: aerobic continuous and interval training; amateur male swimmers; autonomic nervous system function; endurance exercise performance; hemodynamic function; hypoxia 1. Introduction Endurance exercise performance is highly correlated with various physiological components that can be altered by diversiform training methods under hypoxia, includ- ing hematological changes such as erythropoiesis and nonhematological changes such as exercise economy, hemodynamic function, capillary density, and acid-base response in the skeletal muscle [1,2]. Accordingly, exercise training under hypoxia has been used worldwide for decades to enhance endurance exercise performance [3–5]. Hypoxic training can reinforce endurance exercise performance by using three train- ing methods. Living high training high (LHTH) was the rst hypoxic training design to living and training design at 1500–4000 m in the natural altitude conditions that im- prove hematological function, including erythropoiesis and oxygen delivery capacity [6,7]. Int. J. Environ. Res. Public Health2021,18, 3944.

Int. J. Environ. Res. Public Health2021,18, 3944 2 of 13 However, LHTH has a major limitation in that it fails to achieve training of the same inten- sities (e.g., exercise speed and load) as training in a normoxic condition [8]. To overcome these shortcomings of LHTH, living high training low (LHTL), living high at 2000–3000 m and simultaneously training low at below 1500 m, was developed by Dr. Benjamin Levine and James Stray-Gundersen of the United States in the early 1990s [8]. LHTL simulta- neously offers athletes the bene cial effects of hematological function (e.g., improved erythropoiesis) and normoxic training (i.e., maintenance of training intensity) [8,9]. In re- cent years, living low training high (LLTH) has received the most attention from various athletes because it is generally associated with shorter exposure time to hypoxia (approxi- mately three to ve sessions per week of 1–3 h), less effort, less time, and lower cost than LHTH and LHTL [1,10]. LLTH for exercise performance consists of various types of training methods such as continuous hypoxic training (CHT), interval hypoxic training (IHT), sprint interval training under hypoxia (SIH), repeated sprint training under hypoxia (RSH), resistance training under hypoxia (RTH), and voluntary hypoventilation at a low lung volume (VHL) [11]. Among these various LLTH methods (e.g., CHT, IHT, SIH, RSH, RTH, and VHL), CHT and IHT are most commonly used to enhance endurance exercise performance [1,4,5,10]. However, few studies have examined the effects of hypoxic training consisting of CHT and IHT on endurance exercise performance (especially swimming performance) versus contin- uous and interval training in normoxia. Short-term exposure to hypoxia with high-intensity exercise such as CHT, IHT, and CHT + IHT improves endurance exercise performance by enhancing metabolic function, hemodynamic function, and exercise economy [1,5,10,12,13]. However, some studies reported that CHT, IHT, and CHT + IHT did not enhance endurance exercise performance [14–17]. These con icting results are because changes in autonomic nervous system (ANS) function, which are highly correlated with endurance exercise performance, have not been reviewed [10,18–20]. Heart rate variability (HRV) re ects the interaction between the sympathetic nervous system (SNS) and parasympathetic

However, some studies reported that CHT, IHT, and CHT + IHT did not enhance endurance exercise performance [14–17]. These con icting results are because changes in autonomic nervous system (ANS) function, which are highly correlated with endurance exercise performance, have not been reviewed [10,18–20]. Heart rate variability (HRV) re ects the interaction between the sympathetic nervous system (SNS) and parasympathetic nervous system (PNS), which regulate cardiovascular function, and is an effective test method for quantitatively evaluating ANS activity and balance [21]. Dynamic modulation of cardiac and peripheral vascular regulation, including their regulation by the ANS, occurs in response to rapid changes in heart rate (HR) [21,22]. The improvement of HRV function via exercise training is often interpreted as enhanced ANS balance function or vagus nerve activity, which is related to endurance exercise performance [23,24]. Therefore, it is essential to verify the effect of exercise training under hypoxia on changes in HRV related to endurance exercise performance versus normoxia to verify the effectiveness of hypoxic training. However, studies to date of changes in ANS function following hypoxic training are scarce. Therefore, the purpose of this study was to evaluate the effects of aerobic continuous and interval training under hypoxia on hemodynamic function, ANS function, and en- durance exercise performance in amateur male swimmers. We hypothesized that aerobic continuous and interval training under hypoxia would improve endurance exercise per- formance by enhancing hemodynamic and ANS function in amateur male swimmers versus normoxia. 2. Materials and Methods 2.1. Subjects The subjects were competitive amateur male swimmers (n= 20) with no experience in any exercise and training program in normobaric or hypobaric hypoxia (Table). They were equally assigned to the normoxic training group (NTG;n= 10) or hypoxic training group (HTG;n= 10) according to endurance exercise performance and body composition. We ex- plained the experiments and possible side effects to all amateur male swimmers prior to the start of the study and obtained signed consent for participation. The present study was approved by the Institutional Review Board of Konkuk University (7001355-201510-

exercise performance and body composition. We ex- plained the experiments and possible side effects to all amateur male swimmers prior to the start of the study and obtained signed consent for participation. The present study was approved by the Institutional Review Board of Konkuk University (7001355-201510-

Int. J. Environ. Res. Public Health2021,18, 3944 3 of 13 HR-090) in Korea and was conducted in accordance with the provisions of the Declaration of Helsinki. Table 1.Subjects' characteristics. Variable NTG ( n= 10) HTG ( n= 10) pValue Environmental condition (mmHg) Normoxia (760 mmHg) Hypoxia (526 mmHg) - Age (years) 23.90 3.07 24.00 3.06 0.943 Height (cm) 176.3 5.69 176.36 6.04 0.988 Weight (kg) 69.89 13.40 70.18 10.94 0.958 BMI (kg/m 2 ) 23.04 2.88 23.05 1.60 0.992 Percent body fat (%) 23.24 3.88 23.28 3.91 0.982 Values are expressed as the mean standard deviation. BMI, body mass index; HTG, hypoxic training group; NTG, normoxic training group. 2.2. Study Design The present study design is illustrated in Figure. Twenty amateur male swimmers were equally divided into the NTG (n= 10; aerobic continuous and interval training in a normoxia; 760 mmHg) and HTG (n= 10; aerobic continuous and interval training in a hypobaric hypoxia; 526 mmHg; simulated altitude of 3000 m). All testing and training sessions were performed in a 6.5 m wide 7.5 m long 3 m high hypobaric hypoxic chamber (Submersible Systems, Huntington Beach, CA, USA). The temperature within the hypobaric hypoxic chamber was maintained at 20 2 C, and the humidity was maintained at 60 2%.Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 3 of 13 prior to the start of the study and obtained signed consent for participation. The present study was approved by the Institutional Review Board of Konkuk University (7001355- 201510-HR-090) in Korea and was conducted in accordance with the provisions of the Declaration of Helsinki. Table 1. Subjects’ characteristics. Variable NTG ( n = 10) HTG ( n = 10) p Value Environmental condition (mmHg) Normox ia (760 mmHg) Hypoxia (526 mmHg) - Age (years) 23.90 ± 3.07 24.00 ± 3.06 0.943 Height (cm) 176.3 ± 5.69 176.36 ± 6.04 0.988 Weight (kg) 69.89 ± 13.40 70.18 ± 10.94 0.958 BMI (kg/m 2 ) 23.04 ± 2.88 23.05 ± 1.60 0.992 Percent body fat (%) 23.24 ± 3.88 23.28 ± 3.91 0.982 Values are expressed as the mean ±

Hypoxia (526 mmHg) - Age (years) 23.90 ± 3.07 24.00 ± 3.06 0.943 Height (cm) 176.3 ± 5.69 176.36 ± 6.04 0.988 Weight (kg) 69.89 ± 13.40 70.18 ± 10.94 0.958 BMI (kg/m 2 ) 23.04 ± 2.88 23.05 ± 1.60 0.992 Percent body fat (%) 23.24 ± 3.88 23.28 ± 3.91 0.982 Values are expressed as the mean ± standard deviation. BMI, body mass index; HTG, hypoxic training group; NTG, normoxic training group. 2.2. Study Design The present study design is illustrated in Figure 1. Twenty amateur male swimmers were equally divided into the NTG (n = 10; aerobic continuous and interval training in a normoxia; 760 mmHg) and HTG (n = 10; aerobic continuous and interval training in a hypobaric hypoxia; 526 mmHg; simulated altitude of 3000 m). All testing and training sessions were performed in a 6.5 m wide × 7.5 m long × 3 m high hypobaric hypoxic cham- ber (Submersible Systems, Huntington Beach, CA, USA). The temperature within the hy- pobaric hypoxic chamber was maintained at 20 ± 2 °C, and the humidity was maintained at 60 ± 2%. Figure 1. Study design. HR max, maximal heart rate; VO2max, maximal oxygen uptake. The experimental design consisted of the following: a 5-day pre-test period (i.e., 3 testing days and 1 rest day between them), a 6-week training period under each environ- mental condition, and a 5-day post-test period. The post-test period began 3 days after the Figure 1.Study design. HRmax, maximal heart rate; VO 2max, maximal oxygen uptake. The experimental design consisted of the following: a 5-day pre-test period (i.e., 3 test- ing days and 1 rest day between them), a 6-week training period under each environmental condition, and a 5-day post-test period. The post-test period began 3 days after the nal training session. During the 3 days of test sessions, body composition, hematological pa-

Int. J. Environ. Res. Public Health2021,18, 3944 4 of 13 rameters, hemodynamic function, ANS function, and endurance exercise performance were evaluated. On the rst pre- and post-testing days, venous blood samples were collected between 8:00 and 10:00 a.m. after 12 h of fasting to analyze hematological parameters and serum cortisol levels. Thereafter, the body composition and HRV parameters were measured. Subsequently, the maximal oxygen uptake (VO2max) was measured to evaluate the en- durance exercise performance in the afternoon. On the second pre- and post-testing days, hemodynamic function parameters were measured during a 30-min bout of submaximal cycle ergometer exercise. The exercise intensity was set at individual cycle ergometer exer- cise load values corresponding to 70% maximal HR (HRmax) obtained during the pre-test period. On the third testing day, a 400-m time trial record in freestyle was measured by an automatic system installed on an authorized indoor swimming pool (50 m) at sea level in Suwon. During the 6-week training period, 20 amateur male swimmers were equally divided into the NTG (n= 10) and HTG (n= 10), and they performed four kinds of training sessions in each environmental condition (NTG: normoxia, 760 mmHg; HTG: simulated 3000 m hypobaric hypoxia, 526 mmHg) for 90 min: warm-up, aerobic continuous exercise, aerobic interval exercise, and cool-down. The training frequency was 90 min 3 days per week for 6 weeks. Warm-up and cool-down were set at 50% maximum heart rate (HRmax) for each subject for 5 min, then increased by 10% HRmaxevery 5 min and per- formed for 15 min. Continuous aerobic exercise was performed on a treadmill (Precor 932i, Precor, WA, USA) at 75% HRmaxfor 30 min and aerobic interval exercise on a cycle er- gometer (Monark Exercise AB, Vansbro, Sweden) set at the exercise load with 90% HRmax measured in pre-test for 30 min (10 2-min exercise and 1-min rest). The velocity during warm-up, aerobic continuous exercise, and cool-down on a treadmill was changed using an HR monitor (Polar S610i, Helsinki, Finland) to match each HR. Anaerobic interval exercise intensity was set at individual bicycle exercise load values (watts) with 90% HRmax obtained

exercise load with 90% HRmax measured in pre-test for 30 min (10 2-min exercise and 1-min rest). The velocity during warm-up, aerobic continuous exercise, and cool-down on a treadmill was changed using an HR monitor (Polar S610i, Helsinki, Finland) to match each HR. Anaerobic interval exercise intensity was set at individual bicycle exercise load values (watts) with 90% HRmax obtained at pre-test in each environmental condition. In addition, all participants performed equally additional resistance training sessions (three sets of 8–10 repetitions at an exercise intensity range of 70–80% of one-repetition maximum, with 60-s rest per set) in a normoxia composed of a bench press, shoulder press, dumbbell curl, lat pull-down, bent-over-rowing, bent-over-back, push-up, front push, front raise, and bent-over tricep kickback. Additional resistance training frequency was 60 min 3 days per week for 6 weeks. All exercise training sessions in normoxia or hypoxia were supervised by researchers, coaches, and directors. 2.3. Body Composition Body composition parameters (e.g., weight, body mass index, and % body fat) were evaluated after 12 h of fasting using bioelectrical impedance analysis. All participants wore lightweight clothing and were asked to remove any metal items. An Inbody 770 device (Inbody, Seoul, Korea) was used to measure body composition. 2.4. Hematological Parameters Venous blood samples were performed on pre- and post-testing days at rest in nor- moxia to measure hematological parameters. A 5-mL sample of venous blood samples was collected in a heparin tube for whole blood (3-mL) and a serum separation tube for serum (2-mL). To analyze hematological parameters, blood samples were obtained before and after training at rest at normoxia. A 5-mL sample of venous blood was collected in a heparin tube for whole blood (3-mL) and a serum separation tube for serum (2-mL). An XE2100D hematology analyzer (Sysmex, Kobe, Japan) was used to analyze the red blood cell (RBC) count, hemoglobin (Hb) concentration, and hematocrit (Hct). The RBC count and Hct were measured using an impedance-based method. Hb concentration was measured using

XE2100D hematology analyzer (Sysmex, Kobe, Japan) was used to analyze the red blood cell (RBC) count, hemoglobin (Hb) concentration, and hematocrit (Hct). The RBC count and Hct were measured using an impedance-based method. Hb concentration was measured using

Int. J. Environ. Res. Public Health2021,18, 3944 5 of 13 cyanide-free Hb spectrophotometry. Erythropoietin (EPO) levels were measured using an Immulite 2000 XPI analyzer (Siemens, Eschborn, Germany) using the chemiluminescent immunoassay method. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the following formulas: MCV = (Hct/erythrocyte) 10; MCH = (Hb/erythrocyte) 10; and MCHC = (Hb/Hct) 100. 2.5. Hemodynamic Function Parameters Hemodynamic function parameters were evaluated on pre- and post-testing days. All subjects performed a submaximal exercise using a cycle ergometer with a load corre- sponding to 75% HRmax measured before training in normoxia. Oxygen uptake (VO2) was measured using a Vmax-229 breath-by-breath auto metabolism analyzer (SensorMedics, Yorba Linda, CA, USA). HR, stroke volume index (SVi), and cardiac output index (COi) were evaluated noninvasively using a thoracic bioelectrical impedance device (PhysioFlow PF-05, Paris, France). All variables were measured every minute, and the total values were used for HR, VO2, SVi, and COi. 2.6. ANS Function ANS function was assessed by measuring HRV and serum cortisol levels. After approxi- mately10 min of rest, four pads were placed on the wrists and ankles using an HRV meter (LAXTHA; CANS-3000, Daejeon, Korea), and all male amateur swimmers' HRV was evalu- ated in the resting condition. The following parameters were measured: standard deviation of successive differences (SDNN) and root mean square of successive differences (RMSSD) for the time domain methods and total power (TP), low-frequency (LF), high-frequency (HF), and LH/HF ratio for the frequency domain methods. Serum cortisol levels were determined using radioimmunoassay (Coat-A-count; Siemens, Eschborn, Germany). 2.7. Endurance Exercise Performance To evaluate exercise performance, VO2maxwas measured before and after training using the BRUCE protocol for graded exercise testing on a treadmill (Precor 932i) with a Vmax-229 breath-by-breath auto metabolism analyzer (SensorMedics) under normoxia. The 400-m time trial records in freestyle were measured twice by an automatic system installed on an authorized indoor swimming pool (50 m) at sea level in Suwon, and the average time was used. 2.8. Statistical Analysis All statistical analyses were conducted using SPSS version 25.0 (IBM Corp., Armonk, NY, USA)for Windows.

Vmax-229 breath-by-breath auto metabolism analyzer (SensorMedics) under normoxia. The 400-m time trial records in freestyle were measured twice by an automatic system installed on an authorized indoor swimming pool (50 m) at sea level in Suwon, and the average time was used. 2.8. Statistical Analysis All statistical analyses were conducted using SPSS version 25.0 (IBM Corp., Armonk, NY, USA)for Windows. Data are presented as mean standard deviation. The normality of the distribution of all outcome variables was veri ed using the Shapiro–Wilk test. A two-way analysis (time group) of variance with repeated measures of the “time” factor was used to analyze the effects of training programs on each dependent variable. Partial eta-squared ( 2) values were calculated as measures of the effect size. If a signi cant interaction effect was found, a Bonferroni post hoc test was used to identify intragroup changes over time. Additionally, the paired t-test was used to compare the pre- and post- training values of dependent variables in each group separately. An a priori power analysis was performed with G-power for the hemodynamic function parameter (VO2during submaximal exercise) based on previous research [1], indicating that a sample size of 16 participants (8 participants per group) would be required to provide 88% power at an -level of 0.05. We anticipated a more than 10% dropout rate and aimed for a starting population of 20. The level of signi cance was set a priori atp< 0.05.

This study investigates the effects of hypoxic training on endurance performance in amateur male swimmers.