ACUTE PHYSIOLOGICAL RESPONSES TO “RECOVERY INTERMITTENT HYPOXIA” IN HIIT

Introduction: Traditional intermittent hypoxia training improves sport performance after short periods of exposure, but acute exposure to intermittent hypoxia leads to decreased training intensity and technical quality. The solution to overcome these negative effects may be to perform efforts in normoxia and the intervals between efforts in hypoxia, maintaining the quality of training and the benefits of hypoxia. Objective: This study aimed to evaluate the acute physiological responses to hypoxia exposure during recovery between high intensity efforts. Materials and methods: Randomized, one-blind, placebo-controlled study. Sixteen men performed a graded exercise test to determine their maximal intensity and two sessions of high-intensity interval training. The training intervals could be in hypoxia (HRT), FIO2: 0.136 or normoxia (NRT), FIO2: 0.209. During the two-minute interval between the ten one-minute efforts, peripheral oxygen saturation (SpO 2 ), heart rate (HR), blood lactate ([La]), blood glucose ([Glu]) were constantly measured. Results: There were differences in HR (TRN = 120 ± 14 bpm; TRH = 129 ± 13 bpm, p < 0.01) and SpO 2 (TRN = 96.9 ± 1.0%; TRH = 86.2 ± 3.5%, p < 0.01). No differences in [La] and [Glu] TRN (4.4 ± 1.7 mmol.l-1; 3.9 ± 0.5 mmol.l-1) and TRH (5.2 ± 2.0 mmol.l-1; 4.0 ± 0.8 mmol.l-1, p = 0.17). Conclusion: The possibility of including hypoxia only in the recovery intervals as an additional stimulus to the training, without decreasing the quality of the training, was evidenced. Level of Evidence II; Randomized Clinical Trial of Minor Quality.


INTRODUCTION
2][3] The use of IHT as an additional stimulus for training has been a common practice in sports training in different cyclic modalities. 3tudies show the efficacy of IHT in improving sports performance in short periods (i.e.3-5 weeks of training), providing evidence of anaerobic improvements, 4 changes in pH, greater carbonic anhydrase, glucose transport, and HIF1α activity into muscle. 3][6][7] Despite IHT being efficient in promoting positive adaptations, performing training in hypoxia cause a reduction in training intensity and loss of movement quality, reducing the capacity to generate power and decrease the number of efforts performed.[10] IHT results in a paradoxical effect, it can promote additional adaptations due to hypoxia exposure; on other hand, the exposure may decrease the training intensity.The negative effects can "dilute" the possible positive effects on performance. 1 Because of this, performing IHT with efforts at normoxia and pauses between efforts in hypoxia may be an alternative to maintain intensity and technical/mechanical quality during training.This strategy would probably generate positive adaptations from both regular training and hypoxia exposure, avoiding the harmful effects of hypoxia during exercise.However, to the best of our knowledge, no study to date has investigated this possibility under real training conditions.Thus, the present study aimed to assess the acute physiological responses to hypoxic during intervals between high-intensity efforts and compare with normoxia.Our hypothesis was that method could generate physiological alterations on traditional exercise markers as [La], HR and rate of perceived efforts (RPE) great than in normoxia training without decrease the number of efforts performed.

Participants
Sixteen men participated in this study (age= 23±4 years, height= 180±10 cm, weight= 78.4±11.9kg).The participants signed a consent form agreeing to participate in the study.All procedures adopted by the study comply with the university's human research ethics committee (CAAE: 32220020.0.0000.5659)and the Helsinki declaration.

Experimental Design
The participants came to the laboratory on 3 different days, with a minimum 48-hour interval.They were advised to maintain the same diet, to eat regular meals at least two hours before the interventions, and to avoid caffeine/alcohol.All procedures were performed at the same time of day.The laboratory was located at an altitude of 531 meters above sea level, with a controlled temperature of 22 °C.None of the participants had traveled to altitudes above 1,500 m in the previous 6 months.All experiments were performed on a treadmill (Super ATL, Imbrahmed, Brazil).
On the first day, height and body weight were measured, the participants were familiarized with the treadmill and the unidirectional face mask used during the study.Before each intervention, a 5-minute treadmill warm-up was performed at an intensity of 7 km.h - . The subjects performed a graded exercise test (GXT) until exhaustion, to estimate the peak speed achieved (V PEAK ).On the other two days, the participants realized a repeated high-intensity effort training session, with recovery between stimuli being performed in normoxia (T R N) or hypoxia (T R H).These executions were carried out at random, with the participants unaware of the situation they were being exposed to.

Graded Exercise Test (GXT)
The GXT started at 8 km.h -1 , increments of 1 km.h -1 every 3 minutes, until voluntary exhaustion.Between each stage, there was a 30-second interval to collect blood samples and record HR and RPE.The following exhaustion criteria was adopted: a) [La] > 8.0 mmol.l - ; b) maximum HR predicted by age (HRpred = 208 -(0.7 * age); c) RPE > 9 on an RPE scale from 0-10 points. 11he V PEAK was the highest velocity achieved in the GXT; when exhaustion occurred before completing the stage.V PEAK was adjusted using the equation: 12 V PEAK = SLC + (TI/TE) * I Where SLC is the last complete stage speed; TI is the time performed in the last stage; TE is the stipulated time per stage; I was the increase in intensity.

Hypoxia Instrumentation
An Air San unidirectional mask (Air Safety, Brazil) was connected to a 3-meter flexible hose (IVPU, air vacuum PU 1.1/2), the opposite end was connected to a tent (CAT, TentTM, USA) with air capacity of 12,000 liters, connected to a hypoxia generator (CAT-430TM, Altitude Control Technologies, USA).The system remained online throughout the experiment.The F I O 2 selected to hypoxia was of 0.135.The FIO 2 was monitored using an O 2 sensor (Oxygen Senor R-17MED, Teledyne Analytical Instruments, USA).The participants performed the same procedures for the normoxic, however received ambient air (F I O 2 : 0.209).

Training Session
Both training sessions with recovery in normoxia (T R N) and hypoxia (T R H) consisted of ten 1-minute efforts at 100% V PEAK , with 2-minute of passive pauses.In both sessions, the participants carried out their efforts exclusively in normoxia.However, during the intervals, the participants breathed air with either 0.209 or 0.136 F I O 2 .RPE, [La], [Glu] were measured after the end of each effort, the HR, and SpO 2 , were collected each ten seconds, until 120 seconds of interval.(Figure 1)

Hypoxic Dose Calculation (HD)
The HD was estimated as the exposure time in hours (t) multiplied by the reduction in SpO 2 , assuming a resting SpO 2 value of 98%.Thus, HD = (98 -SpO 2 ) x t. 13

Calculation of the Arterial Partial Pressure of Oxygen (PaO 2 )
The SpO 2 values were converted to PaO 2 values using the equation: 14

Blood Sample Analysis
The blood samples were collected using heparinized and calibrated capillary tubes to collect 25μl of blood from the earlobe.The samples were dropped into Eppendorf tubes containing 50μl of 1% sodium fluoride.These tubes were frozen at -20°C for posterior analysis in a blood analyzer to determine [La] and [Glu] (YLS, model 2700, Ohio, USA).

Statistical Analysis
Data normality, homogeneity, sphericity were checked using the Shapiro-Wilk test, Levene test, and Mauchy test, respectively.The Greenhouse-Gesser correction was applied when necessary.The data were expressed in mean ± SD.To compare two means the Student t-test was used for dependent samples.To compare normoxia and hypoxia in different periods the repeated measures ANOVA was used.In the case of significant F values, the Sidak post-hoc test was used.The effect size (ES) and its 95% confidence interval (C.I.) was interpreted following: trivial <0.2, small 0.2-0.6,moderate 0.6-1.2,large 1.2-2.0,and very large >2.0.Only the RPE variable was analyzed using the Wilcoxon signed rank non-parametric f test, considering the value of the rank-biserial correlation (r b ) as the ES.A p-value of 0.05 was used for all tests.The JASP software (version 0.13.1.0,Amsterdam, Netherlands) was employed.The HR responses during the recovery are presented in Figure 4.The mean HR of T R N (120 ± 14 bpm) was lower than that of T R H (129.1 ± 12.7 bpm, p < 0.01, ES= 1.35, C.I.= 0.65, 2.02).

DISCUSSION
The main finding was that training session with hypoxia during the intervals between high-intensity efforts was efficient in causing acute physiological changes in SpO 2 and HR levels compared to normoxia.Thus, the inclusion of hypoxia represents an additional training stimulus, confirming our hypothesis.It is important to highlight that this intervention did not alter the physical efforts, which were all performed in normoxia.The manipulation of the PaO 2 conditions only occurred in the intervals between the maximum 1-minute stimuli, thus not impairing the evaluated individual's motor gestures.
We are unaware of any other studies that investigate the effect of intermittent hypoxia only in the intervals.There is only one study that verifies the effect of hypoxia exposure during recovery after a training session. 15The aforementioned authors applied a continuous submaximal training and used a 30-minute recovery after the end of the exercise. 15hus, we consider the data original.
Hypoxia decreases body oxygen supply, causing acute changes such as decrease in alveolar pressure, PaO 2 , capillary pressure, intramuscular oxygen pressure (PiO 2 ). 16,17 reduction in PiO 2 below to 8.00 mmHg is sufficient to stabilize HIF1α. 18In normoxic, the PHD hydroxylates HIF1α, enabling coupling with the von Hippel-Lindau protein and marking the E3 ubiquitin ligase complex, for degradation of HIF1α in the 265 proteasomes.With the decrease in PHD due to the decrease in PiO 2 , HIF1α stabilizes, increasing its concentration inside the cell's cytosol, causing the MAPK protein to phosphorylate HIF1α.This protein translocates to the cell nucleus, connecting with HIF1β, forming the HIF1α/HIF1β complex.][21] A study using 0.100 F I O 2 , which is lower than the level used in this study (F I O 2 : 0.136), the PaO 2 values at rest were 46 ± mmHg, with respective PiO 2 values of 23 ± 6 mmHg. 16These values were above what was necessary for HIF1α stabilization, indicating that passive hypoxia does not leads to adaptations in the HIF-1α pathway.In corroboration, a review showed that only passive hypoxia was not efficient in causing aerobic or anaerobic changes.

AUTHORS' CONTRIBUTIONS:
All authors contributed individually and significantly to: a) Substantial contribution in the conception or design of the work, acquisition, analysis or interpretation of data for the work -Writing of the work or critical review of its intellectual content; b) Final approval of the version of the manuscript to be published; c) Agree to be held accountable for all aspects of the work, in order to ensure that any matter relating to the completeness or accuracy of any part of the work is properly investigated and resolved.YFF, CDC, FAR, JCA, GLP, DRB, FBMG and MP.
Other studies show that physical exercise in normoxia performed at high-intensity can decrease PiO 2 values from 35 mmHg to 3.1 mmHg, thus exercise at high-intensity seems to be enough to stabilize this pathway. 16,17,23tudies report PaO 2 values of 46 mmHg and PiO 2 values of 2.3 mmHg when high-intensity exercises are combined with hypoxia, being enough for HIF1α stabilization. 17,18In the present study, when hypoxia was added during the recovery intervals, we found PaO 2 values of 50.26 ± 4.57 mmHg.
We are unable to say whether the decrease in oxygen supply in the proposed session was sufficient to stabilize HIFα, since we did not perform muscle biopsies to determine its concentration or expression.The mean SpO 2 value during the intervals was similar to or lower than observed in studies that have demonstrated improved performance with IHT, compared to training performed in normoxia. 3,6,24Based on the aforementioned information, it can be speculated that the hypoxia addition during interval is an additional stimulus to optimize physiological gains, with the advantage of maintaining the "quality of effort, " since the efforts can be performed in normoxia.
The HD was higher in the recovery periods in hypoxia, however, literature provides limited information concerning the HD required to trigger positive adaptations. 25The HD for a given activity seems to be dependent on both exposure time and the variation in SpO 2 values.This variation seems to be influenced by several factors, such as age, training level, and ability to maintain oxygen uptake at altitude and during the ventilatory response to hypoxia.The ventilatory response can alter the entire oxygen consumption cascade from alveoli to muscle PiO 2.

16,26
There was a tendency for [La] to increase in T R H compared to T R N, however no differences were found.This was similar to observed in IHT. 5 The increase in [La] due to hypoxia can be explained in part, by the greater use of anaerobic pathways to generate energy, since the aerobic pathway is compromised when less oxygen is transported to cells. 27In this study, the [Glu] maintained stable during training, suggesting that hypoxia only in the recovery was not enough to increase the lactic demand to generate energy.However, some authors have reported no changes in [La] after hypoxia, a phenomenon known as the "lactate paradox". 28ypoxia can cause reduction in [Glu] accelerating the fatigue and decreasing performance. 29During this study, the participants performed efforts without any decrease in intensity or increase other parameters associated to fatigue.
The HR was frequently used to determine internal training load, it is noted that T R H was effective in changing HR during recovery, showing a greater internal load for T R N, Internal load manipulation was associated with improved performance. 30Thus, add hypoxia in training seems to be alternative to raise internal load, without increase the external load.This hypothesis still needs to be confirmed in future chronic studies.Besides, studies that manipulate training loads during hypoxia have consistently reported additional benefits in performance-related parameters. 4,5he present study has limitations: the subjects did not carry out a training session entirely in hypoxia or other combinations, although our objective was to prevent the entire session from being performed with low O 2 availability for the reasons previously reported.No muscle biopsy was performed to determine biomolecular parameters.

CONCLUSION
We conclude that hypoxia during the intervals between efforts can be an additional and innovative stimulus for high-intensity interval training, without hindering the execution of physical efforts and external training load, maintaining the intensity of efforts, furthermore, this acute model proved to be effective in promoting substantial acute physiological alterations.

Figure 1 .
Figure 1.Representation of the dynamics of the high-intensity intermittent training session with recovery intervals in hypoxia or normoxia.VPEAK -maximum speed reached in the incremental test; E -effort period; R-the interval between efforts; RPE-the subjective perception of effort; SpO2-partial pressure of oxygen, ♥ represents a measurement of heart rate; represents a measurement of blood lactate concentration.

Figure 2 .
Figure 2. Comparison of the average blood oxygen saturation behavior during the recovery intervals, in the different training sessions; panel A -The comparison between the SpO2 average from each series, panel B -SpO2 behavior during the 120 seconds interval.▄ represents training performed with intervals in hypoxia; ○ represents training performed with intervals in normoxia.* means the statistical difference from normoxia with hypoxia situations (p < 0.05).

Figure 3 .
Figure 3.Comparison between arterial oxygen pressure (PaO2) in interval training performed with intervals in hypoxia (TRH) and interval training performed in normoxia (TRN) situation of exposure to hypoxia at rest and situation of hypoxia with high--intensity interval training.* represents statistical difference (p<0.05).

Figure 5 .
Figure 5.Comparison between blood lactate and glucose concentrations during the ten 1min efforts in the different training sessions.White symbols represent normoxia situation; black symbols represent hypoxia situation.

Figure 4 .
Figure 4. Panel A-Comparison between the average frequency during the intervals between efforts.Panel B-Comparison of heart rate behavior during recovery time.Represents training session in normoxia with recovery between intervals in hypoxia; □ represents the training session held in normoxia at maximum intensity.* represents the statistical difference between situations (p<0.05).▄ represents training performed with intervals in hypoxia; ○ represents training performed with intervals in normoxia.