Comparison of systemic and peripheral responses during high-intensity interval exercise under voluntary hypoventilation vs. hypoxic conditions

Participants

Ten healthy physically active males, who practiced recreational physical activity at least 2 days per week, were enrolled in the study (age [mean ± standard error]: 21.8 ± 0.4 years, height: 168.6 ± 1.5 cm, weight: 65.0 ± 2.2 kg). All participants resided at the sea level and none had been exposed to a different altitude as a training intervention. Moreover, none of the participants had any experiences with hypoventilation training before the present study. All participants were provided with an experimental overview and the possible risks of the study were delineated. Written informed consent was obtained from all the patients. This study was approved by the Research Ethics Committee of Ritsumeikan University (BKC2020-079) and was conducted in accordance with the Declaration of Helsinki.

Experimental overview

All the participants visited the laboratory five times during the experimental period. During the first visit, maximal oxygen uptake (VO_2max) was assessed using a graded power test on a cycle ergometer with electromagnetic brake (Power Max VIII; Konami Corp., Tokyo, Japan). During the second visit, a familiarization session for HIIE with VHL was conducted. During the third to fifth visits, each participant performed the main trials, completing a single session of HIIE under three different conditions: normoxia with normal breathing (NOR: 23 °C, 20.9% of fraction of inspired oxygen [FiO₂]), hypoxia with normal breathing (HYP: 23 °C, 14.5% FiO₂), and normoxia with VHL (VHL: 23 °C, 20.9% FiO₂). A randomized crossover design was used. Each trial was separated by at least two days. The trials were performed at the same time (between 13:00 and 15:00) of the day. All trials, including the familiarization session, were completed in a large (14.8 m² ) normobaric hypoxic chamber (FCC-5000S; Fuji Medical Science, Chiba, Japan). The oxygen and carbon dioxide concentrations in the chamber were monitored continuously. Normobaric hypoxia was achieved under HYP conditions by insufflation of nitrogen into the chamber. SpO₂ was monitored during the exercise and the 16-s recovery using a finger pulse oximeter (ATP-W03; Fukuda Denshi, Tokyo, Japan) placed on the tip of the left forefinger. Power output, heart rate (HR), and muscle oxygenation of the vastus lateralis muscle were monitored during the exercise and the 16-s recovery. Blood samples were collected to determine changes in blood glucose and lactate concentrations after each of the three exercise sets, and exercise-related sensations were evaluated simultaneously. The mBF of the vastus lateralis muscle was measured using venous occlusion and near-infrared spectroscopy (NIRS) immediately after exercise completion [19,20].

Exercise protocol

The HIIE was performed using a cycle ergometer with electromagnetic brake (Power Max VIII; Konami Corp.). The pedalling frequency was 100 rpm throughout the exercise period. Additionally, pedaling frequency was fixed using a metronome. The participants were warmed up before the main trials. The warm-up exercise included submaximal pedaling at 50% of VO_2max and subsequent HIIE (3 × 8-s pedaling at 100% of VO_2max). A 16-s recovery at 30% VO_2max was provided between sprints. After the warm-up, the participants commenced the HIIE (three sets of 6 × 8-s pedaling at 170% of VO_2max), with a 16-s recovery at 30% of VO_2max between sprints and a 3-min passive rest period between sets (Figure 1). This exercise intensity (170% of VO_2max) has been utilized in previous studies of HIIE [21]. A previous study using 170% of VO_2max presented marked improvements in both anaerobic and aerobic capacities [21].

Figure 1.

Protocol overview.

The participants underwent HIIE with VHL, except for the recovery period between sprints, which was performed with normal breathing. The procedure of HIIE or repeated sprint exercise with VHL has been described in detail previously [10-12]. Briefly, the participants were required to start each sprint with a first exhalation down to the functional residual capacity, then to hold their breath until the end of the 8-s sprint exercise, and finally to perform a second exhalation down to the residual volume to evacuate the carbon dioxide accumulated in the lungs [11,12,22]. In a familiarization session, SpO₂ was monitored to confirm whether HIIE with VHL could successfully lower SpO₂, as previously reported [10,11].

Maximal oxygen uptake

The VO_2max was assessed at the first visit using a graded power test. Before the test, the participants performed a warm-up session of 5 minutes of pedaling at 100 W. The test began at 70 W, and the load was increased progressively in 30 W increments every 2 minutes until voluntary exhaustion (100 rpm). Respiratory gases were collected and analyzed using an automatic gas analyzer (AE-300S; Minato Medical Science Co., Ltd., Tokyo, Japan). The data were averaged every 30 s. HR was measured continuously during exercise using a wireless HR monitor (RCX5; Polar Electro, Tokyo, Japan). The rating of perceived exertion (RPE) was determined every 2 minutes using a 10-point scale [23].

Measurements during the main trial

During HIIE, the peak and mean power outputs were evaluated for each 8-s sprint under all conditions. SpO₂ was recorded continuously during the 8-s exercise and the 16-s recovery (every second) with a pulse oximeter on the left forefinger (ATP-W03; Fukuda Denshi). HR was recorded continuously during the exercise and the 16-s recovery (every second) using a wireless HR monitor (RCX5; Polar Electro). SpO₂ was presented as the minimum value during the exercise and the 16-s recovery. HR was presented as the average value for all sprints.

Muscle oxygenation variables (oxygenated hemoglobin [oxy-Hb], deoxygenated hemoglobin [deoxy-Hb], total hemoglobin [total-Hb], and tissue oxygen saturation [StO₂]) were evaluated by NIRS (Hb14; ASTEM Co., Ltd., Kanagawa, Japan) [24] in the vastus lateralis muscle at a distance of 50% from the greater trochanter and lateral condyle of the femur. To determine muscle oxygenation variables, the NIRS probe was placed on the muscle at an inter-optode distance of 30 mm. All NIRS variables were measured before (during 1 minute of rest before warm-up under normoxic conditions with participants in a supine position) and during each 16-s recovery period (averaged values). During exercise with VHL, the maximum and minimum values of SpO₂ were expected to appear for a few seconds following the end of the previous sprint [11]. Therefore, the data during the recovery periods were analyzed. All signals were recorded at a sampling frequency of 10 Hz. NIRS data during the exercise and the 16-s recovery under each condition were expressed as relative values to resting levels (before exercise).

To evaluate mBF, venous occlusion was performed on the right leg (most proximal part) using an automatic rapid cuff inflation system (E20; Hokanson, Inc., Bellevue, WA, USA) and a 10-cm wide cuff (SC10D; Hokanson, Inc.) with participants in the supine position. The mBF was evaluated before the warm-up and 40 seconds immediately after exercise completion. This procedure was used in a previous study [24]. Venous occlusion (70–100 mmHg) was applied for 20 seconds to restrict venous outflow. The mBF was calculated in milliliters per minute per 100 g of muscle tissue (mL/min/100 g) from the linear increase in total-Hb during venous occlusion (monitored using NIRS) [20,25-27].

Blood samples were collected from the fingertips before the warm-up (in each condition) and immediately after each set of exercises. Blood glucose and lactate concentrations were immediately measured using a glucose analyzer (Free Style; Nipro Corp., Osaka, Japan) and lactate analyzer (Lactate Pro; Arkray Inc., Kyoto, Japan), respectively.

Perceived breathing difficulty (RPE_breath) and lower limb discomfort (RPE_leg) were rated immediately after each set using a 10-point scale [23].

Statistical analysis

All statistical analyses were performed using the SPSS software (ver. 27.0; IBM Corp., Armonk, NY, USA). All data were expressed as means ± standard errors. Two-way repeated-measures analysis of variance (ANOVA) was used to assess the interaction and main effects of condition and time. A one-way repeated-measures ANOVA was conducted to evaluate the main effect of the condition on the average power output, HR, and SpO₂. Significant interactions and main effects were further analyzed using the Tukey-Kramer post-hoc test. Statistical significance was set at p < 0.05.

SpO₂ and HR

The average values of the minimum SpO₂ during the exercise and the 16-s recovery were 94.9 ± 0.4% in the NOR group, 90.4 ± 0.5% in the VHL group, and 82.8 ± 1.2% in the HYP group. SpO₂ in the VHL group was significantly lower than that in the NOR group (p < 0.001, Figure 2). However, this parameter in the VHL group was significantly higher than that in the HYP group (p < 0.001). The maximum and mean HR in the HYP group were significantly higher than those in the VHL and NOR groups (p = 0.005).

Figure 2.

Changes in minimum SpO₂ during the exercise and the 16-s recovery between sprints. Values were presented as means ± standard errors. ^†p < 0.05, comparison between the VHL and HYP groups . ^‡p < 0.05, comparison between the VHL and NOR groups. ^§p < 0.05, comparison between the HYP and NOR groups.

Power output

The peak power output did not differ significantly among three conditions (Table 1). VHL group presented a slightly (< 1.9%) but significantly lower mean power output than HYP and NOR groups (p = 0.043).

Table 1.

Peak power output and mean power output during exercise.

Blood variables

The blood lactate concentration increased significantly after exercise in all conditions (p < 0.001). Additionally, post-exercise blood lactate concentrations were significantly higher in the HYP group than those in the VHL and NOR groups (p = 0.001). The blood glucose concentration did not differ significantly among three conditions (p > 0.05, Table 2).

Table 2.

Blood glucose and blood lactate concentrations during each exercise set.

Muscle oxygenation

The oxy-Hb level decreased significantly during the 16-s recovery period in the HYP group (p < 0.001, Figure 3). While the oxy-Hb level in the HYP group was significantly lower than that of the NOR group (p = 0.032), there was no significant difference between the VHL and NOR groups (p > 0.05). The deoxy-Hb level increased significantly during the 16-s recovery period under all conditions (p < 0.001, Figure 3). However, no significant difference was observed among three conditions (p > 0.05). The total-Hb level increased significantly during the 16-s recovery period under all three conditions (p < 0.001, Figure 3). However, no significant difference was observed among three conditions (p > 0.05). StO₂ decreased significantly during the 16-s recovery period under all conditions (p < 0.001, Figure 3). Furthermore, StO₂ in the HYP group was significantly lower than those in the VHL and NOR groups (p = 0.024). However, there was no significant difference between this parameter in the VHL and NOR groups (p > 0.05).

Figure 3.

Changes in oxy-hemoglobin (A), deoxy-hemoglobin (B), total-hemoglobin (C), and tissue oxygen saturation (D) during the 16-s recovery between sprints. Values were presented as means ± standard errors. ^*p < 0.05, comparison with the pre-exercise condition (Pre). ^†p < 0.05, comparison between the VHL and HYP groups. ^§p < 0.05, comparison between the HYP and NOR groups.

Muscle blood flow

The mBF increased significantly immediately after exercise under all three conditions (p = 0.002, Figure 4). However, no significant difference was found among three conditions (p > 0.05).

Figure 4.

Changes in mBF. Values were presented as means ± standard errors. ^*p < 0.05, comparison with the pre-exercise condition (Pre).

Perceptual responses

RPE_breath and RPE_leg increased significantly with exercise under all conditions (p < 0.001, Table 3). The average value of RPE_breath in the VHL group was significantly higher than those in the HYP and NOR groups (p < 0.001). In contrast, RPE_leg in the HYP group was higher than that of the VHL group during exercise set 2 (p = 0.032).

Table 3.

RPE for respiration and lower limbs during each exercise set.

Systemic and peripheral variables

As expected, SpO₂ during the exercise and the 16-s recovery in the VHL group was significantly lower than that of the NOR group. Additionally, the decrease in SpO₂ under the VHL condition was comparable to that reported in previous studies [10,11]. Breath holding promotes hypoxia in the body. During exercise with VHL, CO₂ output was inhibited, and CO₂ accumulated in the body due to breath holding [28], leading to an increase in its partial pressure in the blood. Moreover, the increased partial pressure of CO₂ shifted the oxygen dissociation curve to the right, which decreased the affinity of hemoglobin for oxygen, leading to a lower SpO₂ (i.e., the Bohr effect). Although we were unable to determine the partial pressure of CO₂, Woorons et al. [16] reported that exercise with VHL increased the partial pressure of CO₂ compared with the same exercise using normal breathing. Thus, exercise with VHL would facilitate hypoxemia due to the augmented partial pressure of CO₂, which may lower blood and muscle pH. Moreover, the exercise-induced decrease in SpO₂ promoted compensatory vasodilatation by augmenting NO production [29]. Yamaguchi et al. [24] reported a higher muscle blood volume during repeated sprint exercises during hypoxia than during normoxia. Increased muscle blood volume augments the shear stress of vascular endothelial cells, leading to the enhanced production of vascular endothelial growth factor (VEGF) [30]. As VEGF promotes angiogenesis, these physiological responses may improve the oxygen supply to the working muscles and the clearance of metabolites during high-intensity exercise [31-33].

While the exercise-induced increase in blood lactate concentration was greater in the HYP group than in the VHL and NOR groups, no significant difference was found between the latter two. Several studies demonstrated that post-exercise blood lactate concentrations were higher in RSH compared to the same exercise under normoxia [33-35]. In addition, augmented blood lactate concentration after exercise with VHL (versus the same exercise with normal breathing) has been previously reported [28,36] with some inconsistencies [11]. Further investigation is required to compare the exercise-induced acid-base disturbance between repeated sprint exercise with VHL and the same exercise with normal breathing under an equivalent power output.

Muscle blood flow

The decrease in SpO₂ during HIIE with VHL was unstable, and fluctuations in SpO₂ have also been observed previously [37]. The breath holding during VHL only occurred during the exercise, not during the rest periods between sprints, creating an intermittent decrease in SpO₂. Moreover, StO₂ in the HYP group was significantly lower for those in the VHL and NOR groups. However, there was no significant difference between the VHL and NOR groups. In addition, the mBF did not differ significantly immediately after exercise among the three conditions. Therefore, VHL during HIIE did not affect oxidative metabolism in the working muscles. Similarly, Woorons et al. [17] reported that repeated sprint exercises with VHL did not affect muscle tissue oxygenation. Therefore, VHL during repeated sprint exercises had relatively minor impact on local blood flow and oxygen utilization in working muscles. On the other hand, Woorons et al. [38] reported the greater increase in deoxy-Hb during repeated sprint exercise with VHL. Since RSH has been reported to improve oxygen utilization in the fast-twitch muscle fibers [8], the same physiological responses might occur during repeated sprints under hypoxic conditions. Woorons et al. [38] used the VHL method with end-expiratory breath holding held up to the breaking point during exercise and reported the lowest level of SpO₂ among all VHL studies. Therefore, the greater fall in muscle oxygenation might result from severe arterial desaturation. Breath holding for as long as possible (i.e., up to the breaking point) may be a strategy to trigger a decrease in SpO₂ and muscle deoxygenation.

Perceptual variables

The RPE_breath in the VHL group was significantly higher than those in the HYP and NOR groups. Previous studies have suggested that different mechanisms were involved in the improved performance of repeated sprint training with VHL in comparison to RSH11. RSH did not increase oxygen uptake during exercise compared to the same exercise under normoxia [39,40]. However, repeated sprint exercises with VHL were associated with higher oxygen uptake during rest periods between sprints than the same exercise using normal breathing [11]. Since the RPE_breath was higher under VHL condition in the present study, metabolic stress on the respiratory muscles, rather than the vastus lateralis muscle, might increase by utilizing VHL.

The present study had several limitations. We used HIIE (equivalent to 170% of VO_2max) rather than repeated sprinting exercises ( exercise with maximal effort) because we thought that HIIE at fixed exercise intensity with VHL would be applicable, as compared to exercise with maximal effort. However, the mean power output was slightly, but significantly, lower in the VHL condition than in the other two conditions, which may affect the present outcomes. Therefore, a similar comparison is required using repeated sprint exercises with VHL. Moreover, since the decrease in SpO₂ was lower in exercise with VHL than in hypoxic conditions (14.5% FiO₂), a comparison to moderate hypoxia around 16.4% FiO₂ (a simulated altitude of 2,000 m) may be more appropriate.

In conclusion, HIIE-induced SpO₂ decrease was smaller under hypoxic conditions than during VHL. Moreover, mBF was not enhanced by the addition of VHL during repeated sprint exercises.