Sprint Interval Exercise Improves Cognitive Performance Unrelated to Postprandial Glucose Fluctuations at Different Levels of Normobaric Hypoxia

bjective: The aim of our study was to examine cognition response to sprint interval exercise (SIE) against different levels of hypoxia. Research design and methods: 26 recreational active males performed SIE (20 6 s of all-out cycling bouts, 15 s of passive recovery) under normoxia (F IO 2: 0.209), moderate hypoxia (F IO 2: 0.154), and severe hypoxia (F IO 2: 0.112) in a single-blinded crossover design. Cognitive function and blood glucose were assessed before and after 0, 10, 30, and 60 min of the SIE. Heart rate (HR), peripheral oxygen saturation (SpO 2), and ratings of perceived exertion (RPE, the Borg 6–20-point scale) during each SIE trial were recorded before and immediately after every ve cycling bouts, and after 0, 10, 30, and 60 min of the SIE. Results: All the three SIE trials had a signi cantly faster overall reaction time in the Stroop test at 10 min after exercise as compared to that of the baseline value (p= 0.003,h 2=

scale) during each SIE trial were recorded before and immediately after every ve cycling bouts, and after 0, 10, 30, and 60 min of the SIE. Results: All the three SIE trials had a signi cantly faster overall reaction time in the Stroop test at 10 min after exercise as compared to that of the baseline value (p= 0.003,h 2= 0.606), and returned to normal after 60 min. The congruent RT at 10 min after SIE was signi cantly shorter than that of the baseline (p< 0.05, h 2= 0.633), while the incongruent RT at both 10 min and 30 min were signi cantly shorter than that measured at baseline (p< 0.05,h 2 = 0.633). No signi cant differences in terms of accuracy were found across the three trials at any time points (p= 0.446,h 2= 0.415). Blood glucose was signi cantly reduced at 10 min and was sustained for at least 60 min after SIE when compared to pre-exercise in all trials (p< 0.05). Conclusions: Acute SIE improved cognitive function regardless of oxygen conditions, and the sustained improvement following SIE could last for at least 10–30 min and was unaffected by the altered blood glucose level. Keywords:high-intensity interval training; normoxia; executive function; glucose 1. Introduction High-intensity interval training (HIIT) has been promoted over the last decade as an effective form of exercise training for improving cardiovascular and metabolic function and mental health in active young adults [1,2] and patients with coronary artery disease [3]. The bene ts of the lower volume and time commitment of HIIT in overcoming the time- related barrier to physical activity adherence show clear superiority compared to traditional endurance exercise. As such, extreme short (<10 s) bouts of all-out sprint interval exercise (SIE), classi ed as one form of HIIT, seems to be an attractive option for achieving exercise- related health bene ts [4]. Hypoxic training has been widely adopted as an effective strategy to enhance exercise performance and physical tness in athletes for decades. More recently, this training strat- egy has shown its bene cial effects on cardiometabolic health and weight management in untrained

as one form of HIIT, seems to be an attractive option for achieving exercise- related health bene ts [4]. Hypoxic training has been widely adopted as an effective strategy to enhance exercise performance and physical tness in athletes for decades. More recently, this training strat- egy has shown its bene cial effects on cardiometabolic health and weight management in untrained individuals [5–8]. Compared to training at sea level, hypoxic training could further enhance blood oxygen transport capacity and aerobic endurance, as reported in pre- vious studies [8–10]. However, the optimal dose of hypoxic severity for health-enhancing J. Clin. Med.2022,11, 3159.

J. Clin. Med.2022,11, 3159 2 of 14 strategies is yet to be determined in view of the widely varying hypoxic exposure duration and severity used in the experimental protocols in the previous studies. The discrepancies in methodology make generalization challenging. Protocols that elicit bene cial effects on cardiorespiratory, metabolic, and immune health without pathology are more likely to arise from hypoxic exposure to a 9–16% fraction of inspired O2[11]. Under such con- ditions, the exaggerated reduction in oxygen saturation increased ventilatory demand in response to hypoxia as a compensatory effect [12], with a more severe hypoxic level during a short period of exposure most often leading to bene cial cardiovascular effects [11]. This suggested that the severity of hypoxia appears to be the pivotal factor to determine this critical dependency. A number of studies have demonstrated that acute exposure to hypoxia can negatively alter human cognitive function due to reduced oxygen partial pressure [13]. Cognitive dete- rioration may occur in acute hypoxic exposure at altitudes above 6000 m (FiO2: 0.097) [14]. Physical activity has previously been shown to enhance cognitive function [15–17]. It has been found that SIE improved selective attention [18], lexical learning [19], and executive function [20] in adults, and such bene cial effects were greater [19] and lasted longer [20] when compared to those resulting from continuous aerobic exercise. Due to the discrep- ancies in severity and duration of hypoxic exposure, however, results from randomized control trials and reviews investigating exercise combined with hypoxia showed both bene- cial [21–27] and detrimental effects [28–34] on cognitive performance. Notably, studies on cerebral oxygenation have reported that central oxygenation between normoxia and hy- poxic conditions were different. Near-infrared spectroscopy (NIRS) measurement revealed that during incremental exercise under hypoxia, stages in low to high intensity elicit a larger degree of cerebral deoxygenation compared with normoxia, which may limit cognitive activity and exercise performance [35]. Investigations that used electroencephalography and transcranial magnetic stimulation reported that as the severity of the hypoxia increased, cerebral oxygenation became more predominant in in uencing cognitive activity and ex- ercise performance [36]. Our previous studies demonstrated that

low to high intensity elicit a larger degree of cerebral deoxygenation compared with normoxia, which may limit cognitive activity and exercise performance [35]. Investigations that used electroencephalography and transcranial magnetic stimulation reported that as the severity of the hypoxia increased, cerebral oxygenation became more predominant in in uencing cognitive activity and ex- ercise performance [36]. Our previous studies demonstrated that high-intensity exercise under moderate hypoxia (FiO2: 0.154) [34] and moderate-intensity exercise under severe hypoxia (FiO2: 0.12) [25] had no detrimental effect on cognitive function. More information about the impact of different hypoxic severities on cognitive function is needed. Acute SIE improved postprandial glucose metabolism in healthy subjects [37], an obese population [38,39], and type 2 diabetes patients [40] immediately or up to 24 h after exercise. Hypoxia is known to improve glucose effectiveness, and is suggested to facilitate further improvement in insulin sensitivity when combined with exercise [41]. Glucose is the major energy resource for the brain when processing cognition-related information. The brain is sensitive to disruptions in energy supply, whereas a modest increase in blood glucose concentration was associated with better cognitive performance [42]. Research has also shown that dif cult cognitive tasks, such as those involving executive function pertaining to the frontal cortex, were more susceptible to glycemic alteration [43]. Available data indicate that acute hypoglycemia, or when the blood glucose concentration was below 3 mmol L 1 , would impair cognition performance [44]. Therefore, glucose uctuations induced by exercise in hypoxic conditions may have a negative in uence on cognitive performance. Given the above, this study used a single-blinded crossover design to evaluate the effects of SIE under normoxia, moderate hypoxia (FIO2: 0.154, simulating an altitude of 2500 m), and severe hypoxia (FIO2: 0.112, simulating an altitude corresponding to 5000 m) on cognitive function. We particularly focused on whether the cognitive-improvement effects of SIE were impaired while performing under hypoxia as the severity of hypoxia increased, and whether blood glucose was associated with cognitive performance during exercise under hypoxia. Given that brain function and tissue integrity are dependent on a continuous and suf cient oxygen supply, we

corresponding to 5000 m) on cognitive function. We particularly focused on whether the cognitive-improvement effects of SIE were impaired while performing under hypoxia as the severity of hypoxia increased, and whether blood glucose was associated with cognitive performance during exercise under hypoxia. Given that brain function and tissue integrity are dependent on a continuous and suf cient oxygen supply, we hypothesized that cognitive function would be impaired during exercise under hypoxia as the severity of hypoxia increased.

J. Clin. Med.2022,11, 3159 3 of 14 2. Materials and Methods 2.1. Participants The study was approved by the Research Ethics Panel of the University of Macau, and all experimental procedures were in accordance with the declaration of Helsinki. Advertisements including research purposes and inclusion criteria were posted on the e-bulletin board of the university to recruit recreational active males. The inclusion criteria were: (1) a maximal oxygen uptake ( . V O2max) level of over 40 mL kg 1 min 1 but lower than 55 mL kg 1 min 1 ; (2) right-hand dominant; (3) nonhighland resident (above 1000 m); (4) without prior experience in hypoxic training; (5) free of any known neurological, cardiovascular, and pulmonary disorders; and (6) free from color blindness or abnormal vision. Smokers or those taking medication or having any physical barriers to performing SIE under hypoxia were excluded. Volunteers who were interested in this study were required to perform an incremental ramp test in the kinesiology lab to determine their . VO2maxand eligibility. An a priori power analysis was conducted using G*Power Version 3.1 to estimate the sample size. When the effect size was set at medium (f= 0.25), 21 participants would be needed to detect a signi cant difference in a two-way repeated-measures ANOVA with a power of 80% and a signi cance level of 5%. Considering a 20% dropout rate, 26 recreational active males were invited to participate in this study. All participants were informed of the experimental procedures and potential risks, and provided a written consent prior to participation. This study was conducted from October 2019 to September 2020. A total of 20 recreational active males (age: 21.4 2.0 y, stature: 175.9 9.1 cm, body mass:68.7 11.4 kg , body mass index (BMI): 22.1 2.1 kg m 2 , maximal oxygen uptake ( . V O2max): 42.9 1.3 mL kg 1 min 1 ) completed all experimental trials and required measurements (Figure).J. Clin. Med. 2022, 11, x FOR PEER REVIEW 3 of 14 continuous and sufficient oxygen supply, we hypothesized that cognitive function would be impaired during exercise under hypoxia as

index (BMI): 22.1 2.1 kg m 2 , maximal oxygen uptake ( . V O2max): 42.9 1.3 mL kg 1 min 1 ) completed all experimental trials and required measurements (Figure).J. Clin. Med. 2022, 11, x FOR PEER REVIEW 3 of 14 continuous and sufficient oxygen supply, we hypothesized that cognitive function would be impaired during exercise under hypoxia as the severity of hypoxia increased. 2. Materials and Methods 2.1. Participants The study was approved by the Research Ethics Panel of the University of Macau, and all experimental procedures were in accordance with the declaration of Helsinki. Ad- vertisements including research purposes and inclusion criteria were posted on the e-bul- letin board of the university to recruit recreational active males. The inclusion criteria were: (1) a maximal oxygen uptake (V̇O 2max) level of over 40 mL∙kg −1 ∙min −1 but lower than 55 mL∙kg −1 ∙min −1 ; (2) right-hand dominant; (3) nonhighland resident (above 1000 m); (4) without prior experience in hypoxic training; (5) free of any known neurological, cardio- vascular, and pulmonary disorders; and (6) free from color blindness or abnormal vision. Smokers or those taking medication or having any physical barriers to performing SIE under hypoxia were excluded. Volunteers who were interested in this study were re- quired to perform an incremental ramp test in the kinesiology lab to determine their V̇O 2max and eligibility. An a priori power analysis was conducted using G*Power Version 3.1 to estimate the sample size. When the effect size was set at medium (f = 0.25), 21 participants would be needed to detect a significant difference in a two-way repeated-measures ANOVA with a power of 80% and a significance level of 5%. Considering a 20% dropout rate, 26 recrea- tional active males were invited to participate in this study. All participants were in- formed of the experimental procedures and potential risks, and provided a written con- sent prior to participation. This study was conducted from October 2019 to September 2020. A total of 20 recreational active males (age: 21.4 ± 2.0 y, stature: 175.9 ± 9.1 cm, body mass: 68.7 ±

males were invited to participate in this study. All participants were in- formed of the experimental procedures and potential risks, and provided a written con- sent prior to participation. This study was conducted from October 2019 to September 2020. A total of 20 recreational active males (age: 21.4 ± 2.0 y, stature: 175.9 ± 9.1 cm, body mass: 68.7 ± 11.4 kg, body mass index (BMI): 22.1 ± 2.1 kg∙m −2 , maximal oxygen uptake (V̇O 2max): 42.9 ± 1.3 mL∙kg −1 ∙min −1 ) completed all experimental trials and required meas- urements (Figure 1). Figure 1. Flow diagram of the study. 2.2. Experimental Design This study included a familiarization session and three experimental trials. During the familiarization session, participants were familiarized with the experimental proce- dures and practiced the SIE protocol and the Stroop test. After the familiarization session, the participants accomplished three main trials at the same time on different days, namely an SIE trial under normoxia (N), an SIE trial un- der normobaric moderate hypoxia (M, FIO2: 0.154, simulated at an altitude of 2500 m), and Figure 1.Flow diagram of the study. 2.2. Experimental Design This study included a familiarization session and three experimental trials. During the familiarization session, participants were familiarized with the experimental procedures and practiced the SIE protocol and the Stroop test. After the familiarization session, the participants accomplished three main trials at the same time on different days, namely an SIE trial under normoxia (N), an SIE trial under normobaric moderate hypoxia (M, FIO2: 0.154, simulated at an altitude of 2500 m), and an SIE trial under severe hypoxia (S, FIO2: 0.112, simulating an altitude corresponding to 5000 m). The three trials were assigned in a random and counterbalanced order, and were interspersed by three to seven days of washout period.

J. Clin. Med.2022,11, 3159 4 of 14 On the day of trials, participants were required to report to the lab before 17:50 to perform a baseline blood glucose test (using glucose and lactate analyzer, Biosen C-Line, EKF diagnostics, Barleben, Germany) and a Stroop test after a 10 min rest. Then, a standard dinner was provided to them at 18:00. A modi ed gas-mixing system (Everest Summit II Hypoxic Generator, New York, NY, USA) was used to generate normoxic or hypoxic gases, and the normoxic or hypoxic gas mixtures were delivered to participants through a breathing mask and tubes. At 19:30, participants were tted with the breathing mask connected to the gas-mixing system, and they rested on the seat for 25 min. Then, a pre- exercise test of blood glucose and the Stroop test were performed. The SIE session (5 min warmup, 7 min exercise, and 3 min cooldown) began at 20:00 under the conditions of normobaric normoxia (i.e., the N trial) or normobaric hypoxia (i.e., the M and S trials). Stroop tests were carried out subsequent to the measurement of blood glucose levels at 10, 30, and 60 min after SIE. The breathing mask was taken off at 20:30. Participants were unaware of the normoxic or hypoxic condition of the experimental trials, and they were required to guess the oxygen condition of the SIE trial by answering the question “Under what condition do you think you were exercising, normoxia, moderate hypoxia or severe hypoxia (i.e., sea level, 2500 m or 5000 m)?” However, no con rmation was given to the participants in order to avoid a potential in uence on the subsequent trials. 2.3. Sprint Interval Exercise The 7 min SIE trial consisted of 20 repetitions for 6 s of high-intensity cycling bouts interspersed with 15 s of passive recovery. Participants pedaled maximally against a load equivalent to 7.5% of their body mass during the 6 s work durations, and underwent passive recovery during the 15 s rest periods on a cycle ergometer (Monark 839E, Vansbro, Sweden). Heart rate (HR) and peripheral oxygen saturation (SpO2) during each SIE trial were

high-intensity cycling bouts interspersed with 15 s of passive recovery. Participants pedaled maximally against a load equivalent to 7.5% of their body mass during the 6 s work durations, and underwent passive recovery during the 15 s rest periods on a cycle ergometer (Monark 839E, Vansbro, Sweden). Heart rate (HR) and peripheral oxygen saturation (SpO2) during each SIE trial were recorded continuously by a pulse oximeter (Radical-7 Pulse CO-Oximeter, Masimo, Irvine, CA, USA), while ratings of perceived exertion (RPE, the Borg 6–20-point scale) were recorded before and immediately after every ve cycling bouts, as well as 10 min, 30 min, and 60 min after the SIE. Participants were introduced to the scale and given standardized instructions on how to rate their overall sensations of effort (including the feeling of peripheral working muscles and joints, central cardiovascular and respiratory systems, and the central nervous system [45]) before each exercise session. Peak power, average power, and the fatigue index were calculated using Monark Anaerobic Test software (Sports Medicine Industries, Inc., St. Cloud, MN, USA). The fatigue index was analyzed to determine the level of fatigue during the anaerobic exercise [46]. A higher fatigue index indicated a lower ability to maintain anaerobic performance over a series of sprints. The exercise HR was estimated using the average HR during the entire exercise session. The HRmaxwas determined as the highest value attained when the . V O2maxtest was performed during the familiarization session. The percentage of HRmaxwas estimated using the ratio between exercise HR and HRmax. 2.4. Cognitive Test For the cognitive task, the color–word matching Stroop test [47] was adopted to re ect prefrontal cortex function. A laptop with a pre-established E-Prime program was used to administrate the Stroop test. For each trial, two lines of letters were displayed on the laptop screen, and participants were instructed to determine whether the color of the letters on the top line matched the name of the color displayed on the bottom line (Figure), and to input their response by pressing “F” or “J” buttons to provide a “yes” or “no” choice with their index ngers. Each

lines of letters were displayed on the laptop screen, and participants were instructed to determine whether the color of the letters on the top line matched the name of the color displayed on the bottom line (Figure), and to input their response by pressing “F” or “J” buttons to provide a “yes” or “no” choice with their index ngers. Each experimental session consisted of 30 trials, including 10 neutral trials, 10 congruent trials, and 10 incongruent trials, which appeared in a random order. For the neutral trial, the top line contained a group of Xs (XXXX) printed in red, green, blue, or yellow, and the bottom line contained the word “RED”, “GREEN”, “BLUE,” or “YELLOW” printed in black. For the congruent trial, the top line contained the word “RED”, “GREEN”, “BLUE,” or “YELLOW” printed in a congruent color. For

This study evaluates cognitive function improvements from sprint interval exercise under varying hypoxic conditions.