MAXIMUM EFFORT TRAINING PERFORMED IN HYPOXIA ALTERS THE MOOD PROFILE

ABSTRACT Introduction: Physical exercise at high altitude has become constant. However, the risks associated with this type of exercise represent a major concern, considering the influence of important stressors such as hypoxia and physical exercise on psychobiological and physiological responses. Objective: Analyze the mood state and behavior of physiological variables of volunteers subjected to a progressive loading protocol until they reached maximum volitional exhaustion, both at sea level and at a simulated altitude of 4500 meters. Method: For both conditions studied, the volunteers responded to two instruments that assess mood responses: The Brunel Mood Scale and the Visual Analogue Mood Scale. They also underwent blood sampling to measure blood lactate levels and to evaluate oxygen-hemoglobin saturation. These procedures were performed before, immediately after, and 30 and 60 minutes after the end of the protocol. Results: Hypoxia triggered negative effects on mood responses, especially when compared to sea level conditions. An increase in fatigue level (p=0.02) and mental confusion (p=0.04) was observed immediately after the exercise session, and reduction of vigor (p=0.03) was noted at 30 minutes, accompanied by a reduction in oxygen-hemoglobin saturation immediately after the session and at 30 minutes. There was also an increase in blood lactate levels immediately after the session (p=0.006). Conclusion: The particularities of the hypoxic environment associated with maximum exercise are able to cause a deterioration of mood and physiological responses, which can negatively modulate physical performance. This is a cross-sectional clinical study.


INTRODUCTION
Countless people seek adventure and leisure, as well as practice physical exercises (PE) at high altitudes, directly exposing themselves to the consequences of reduced oxygen partial pressure of hypoxia 1 .
Hypoxia represents a threat to homeostasis, as it reduces an organism' s ability to bind, transport, and use oxygen through the combined functions of the respiratory, cardiovascular, and hematological systems 1 .These threats are counteracted by immediate physiological responses that induce physiological alterations, for example variations in heart rate (HR), systolic volume, cardiac output, muscle blood flow, ventilation, use of energy substrates, and mitochondrial function have been observed in the autonomic nervous system 2,3 .
At high altitudes, the maximum capacity of PE, time of resistance, and performance are reduced, in contrast to the increase in general fatigue.Such consequences are related to the combined effects of three important factors: 1) reduction in the partial pressure of oxygen, resulting in a lower supply and utilization of oxygen by the skeletal muscles; 2) reduction of atmospheric density and its important impact on air resistance; and 3) the process of acclimatization to altitude, which in turn can affect oxygen transportation [3][4][5] .
In this context, in addition to the physiological responses of the combined stress of hypoxia and PE psychological capacities such reactiveness, attention, cognitive performance, mental efficiency and mood responses (including anxiety) are also influenced and impaired [6][7][8] .
Lemos et al. 9 observed that men exposed to hypoxia simulated at 4.500m exhibited increased scores in affective dimensions such as depressive mood, anger, and fatigue.In addition, vigor, attention, visual memory, working memory, concentration, executive function, inhibitory control, and mental processing speed deteriorated in this environmental condition.
These losses represent significant risks that are influenced by the cognitive appraisal of physiological responses, and thus, more precisely, mood responses serve as early indicators of the adverse effects of this condition 10 , consistent with Bahrke and Shukitt-Halle 11 that posit that environmental changes tend to influence psychological functioning before physiological functioning.
The association between psychological function and PE in normoxia is well established and the improvement of cognitive behavior and affective experience has been emphasized 12 , encompassing an enhanced psychological well-being with a concomitant reduction of tension, anger, depression, and confusion 13 .
This study aims to elucidate the mood profiles of participants undergoing an PE regime performed under maximum voluntary ventilation and a simulated hypoxia condition and investigate possible associations with physiological factors and contribute to the detection of environmental stress.

MATERIAL AND METHODS
The study involved 12 physically active men, with a mean age of 21.92 ± 2.07 years, height 1.76 ± 0.04 m, total body mass of 70.36 ± 10.83 kg, and body mass index of 22.69 ± 3.42 kg/m 2 .To confirm their cardiovascular health, the participants performed a resting electrocardiogram and an exercise stress test, and those deemed suitable were recruited to the study and participated in a progressive regimen of loads until maximum voluntary ventilation (TEmax), carried over in two experimental conditions, with an interval of 7 days between them: 1) Sea level; 2) Simulated hypoxia at an altitude of 4500 meters.
The study was approved by the Ethics Committee of the Federal University of São Paulo/ São Paulo Hospital (#0620/09).All participants signed an informed consent form.
The tests were performed at the same time on a treadmill (Lifefitness® 9700HR, Schiller Park, IL, USA) with a fixed inclination of 1% in order to simulate physical exercise in open places 14 .The TEmax protocol consisted of speed increments of 1 km/h every minute, with an initial warm-up load of three minutes at 7 km/h.The test ended when a participant reached maximum voluntary ventilation; this was regarded as the incapacity to sustain the speed of the treadmill over 15 seconds, or until the participants request to stop the test even when encouraged verbally.
At sea level condition, ergo-spirometry was performed to determine the following respiratory variables: peak oxygen consumption, oxygen consumption at ventilatory threshold I, oxygen consumption at ventilatory threshold II, maximum HR, HR at ventilatory threshold I, HR at ventilatory threshold II, maximum speed, speed at the intensity of the ventilatory threshold I and speed at the intensity of the ventilatory threshold II.
These variables were methodologically obtained by measuring respiratory gas exchange with a metabolic system (COSMED model Quark PFT -Pulmonary Function Testing -FRC & DLCO, Italy).The system was calibrated prior to the completion of each protocol using a known gas concentration.A Hans Rudolph ® flow-by face mask (Kansas City, MO, USA) was used.
The tests performed in the simulated hypoxia condition were conducted in a normobaric chamber (CAT-Colorado Altitude Training™/CAT-12 Air Unit), consisting of a microprocessor system capable of reading electrical signals from two oxygen sensors, and a carbon dioxide and an atmospheric pressure sensor.The information from these sensors are linearized and used to calculate the simulated altitude, to be able to regulate and control the air units, purifiers, and fans to sustain the desired altitude.
In each experimental condition the participants partook in subjective and physiological assessments.These evaluations were performed before, immediately after, and 30 minutes and 60 minutes after the end of the TEmax.In the tests conducted in the hypoxia condition, participants were evaluated immediately after the experiment, 30 minutes after, at sea level at baseline, and 60 minutes after (Figure 1).

Evaluation of Mood
1. Brunel Mood Scale (BRUMS): Developed to measure mood 15 , this instrument was adapted from the "Profile of Mood States" (POMS) 16 , validated for the Portuguese language 17 .It consists of a list of 24 mood related adjectives, where participants note what they feel in relation to each adjective, using a Likert scale of 0 to 4. 2. Visual analogues of Mood scales (VAMS): Consists of 16 100-mm analog scales that are used to assess changes in participants' mood by marking on a vertical line 18 .

Physiological evaluation
1. Blood lactate: Samples of 25mL of arterial blood were collected from the earlobe through a heparinized capillary.The blood was immediately transferred to 1.5 ml polyethylene microtubules with an Eppendorff-type cap containing 50 μL of 1% Sodium Fluoride (NaF) solution.The samples were analyzed with an electrochemical analyzer (YSI STAT 2300, Yellow Springs, Ohio, USA).

Saturation of Oxy-hemoglobin:
The measurement of oxyhemoglobin saturation was performed using a digital finger Oximeter (Fingertip Pulse ® -Model MD300C202) with a red and infrared optical sensor placed on the fingertip that continuously and non-invasively monitors arterial oxygenation.

Statistical analysis
The statistical analysis was conducted using Statistica ® 12.0 software.The normality of data was verified using the Shapiro-Wilk test.Measures of central tendency were calculated for the descriptive data analysis.A repeated measures ANOVA with the factors TIME and GROUP was used to analyze interactions between results.The Duncan post hoc test was used to detect significant differences.The level of significance was set at p < 0.05.

RESULTS
Table 1 presents the descriptive analysis of the cardiorespiratory variables of TEmax at sea level.
In the hypoxia condition, a decrease in vigor and increased fatigue were observed immediately after (p = 0.00002 both dimensions) and 30 minutes later (vigor: p = 0.0005; fatigue: p = 0.003); participants remained in the normobaric chamber.After 60 minutes, vigor scores (p = 0.04) were higher than baseline values unlike fatigue scores.
The VAMS instrument only indicated differences in relation to time.In both experimental conditions maximum PE was capable of reducing anxiety scores (F (3.66) = 10.83,p<0.05) immediately after the experiment.
An effect of time and group was observed in oxyhemoglobin saturation (Figure 3), (F (3.66) = 22.10, p<0.05).In the hypoxia condition reduced saturation was observed immediately after the experiment (p=0.0001) and 30 minutes later (p=0.00005),when compared to baseline and 60 minutes (p=0.00005), and respective moments in normoxia.Experimental design followed by 12 volunteers undergoing a TEmax regimen in two experimental conditions, sea level and hypoxia simulated at an altitude of 4,500 meters.The experimental preparation of the participants was 15 minutes, allowing participants to be placed and prepared in the chamber for the beginning of the procedure.After these adjustments, the participants commenced the TEmax.Subjective and physiological evaluations were performed at baseline, follow up (immediately after), 30 minutes, and 60 minutes after TEmax, in their respective experimental conditions.

Imed. After
Sea level

Hypoxia
An increase in blood lactate levels was observed immediately after the experiment and 30 minutes later compared to baseline and 60 minutes in both experimental conditions (F (3.51) = 158.73,p<0.05).In addition, in terms of experimental conditions (F (3.51) = 4.25, p<0.05), there was a marked increase immediately after the experiment of this metabolite in hypoxia compared to normoxia (p=0.006)(Figure 4).

DISCUSSION
Hypoxia causes numerous and critical adjustments in mood and physiological variables, that are amplified by the completion of maximum PE.
Robust changes were observed on BRUMS dimensions during hypoxia, including increased fatigue, increased mental confusion, decreased vigor, and higher total mood disturbances.Repeated measures ANOVA with Duncan post hoc tests.Data presented as mean ± standard error, with significant results for p≤0.05. a -significantly different from baseline, in the same condition; b -significantly different from immediately after, in the same condition.OFA -Other feelings and attitudes.

60'After
These findings corroborate a study by Lemos et al. 9 , that observed that the scores BRUMS on depression, anger, and fatigue were higher in individuals exposed to hypoxia compared to normoxia.In turn, vigor scores were lower in hypoxia compared to normoxia.
The losses on the affective dimensions observed in our study are consistent with previous data that emphasizes that hypoxia at altitudes of 4.500m can cause cognitive impairments in attention, short-term memory, arithmetic skills, and decision-making, as well as psychobiological changes, in particular increases in euphoria, irritability, hostility, and neuropsychological impairment 11 .
Furthermore, not only do high altitudes influence mood states, but Li et al. 19 have highlighted the interference of hypoxia (simulated at 2,800m) on mood uncovered during the hour of this condition.These individuals showed a gradual increase in stress and fatigue, and a reduction in vigor, that was aggravated by the gradual ascent to a high altitude.
The affective changes evaluated by the VAMS instrument further support these findings.In hypoxia, a reduction in scores was exhibited in physical sedation, mental sedation, and other feelings and attitudes, particularly immediately and 30 minutes after the experiment indicating an increase in alertness during exposure to hypoxia.
These results corroborate Weiss et al. 20 who described the effects of sub-acute intermittent hypoxia (at an altitude of 3.962m) on various cognitive functions, and in particular alertness.The absence of significant deficits in objective alertness and attention, main findings of this study, can be explained by the reduced availability of oxygen during hypoxia, that plays a role in the synthesis of catecholamines, the primary hormones responsible for increases in alertness 21 .
Moreover, it is noteworthy that there were reduced scores in anxiety in both experimental conditions immediately after the completion of TEmax.These results suggest that despite exposure to hypoxia, maximum PE still exerted an anxiolytic effect 22 ; unlike what was observed after moderate PE in the same condition 23 .Extended exposure to hypoxia, intensity of the effort and within subject variability all serve as plausible explanations for the varied results 3,23 .
The reduced oxyhemoglobin saturation immediately and 30 minutes after exposure to the hypoxia condition are indicative of a return to normoxia and the subsequent normalization of oxyhemoglobin saturation.
In agreement with these findings, Mollard et al 24 found a larger reduction of oxyhemoglobin saturation in the trained group compared to a untrained group that underwent a maximal test of incremental load in a condition of simulated hypoxia at 5 altitudes.One of the objectives of this study was to evaluate whether the reduction of the maximum VO 2 of participants exposed sharply to hypoxia could only be explained by a decrease in oxyhemoglobin saturation or by the behavior of the HR.The study demonstrated that oxyhemoglobin saturation is a strong predictor of maximum VO 2. This is primarily because of the limitations of the alveolar-capillary diffusion that is the outcome of an increase in cardiac output, reducing the transit time of red blood cells in pulmonary capillaries.
Differences between the experimental conditions were found in the variables vigor, fatigue, mental confusion, saturation of oxyhemoglobin, and blood lactate levels highlighting the impact of hypoxia on mood and physiological responses.
The blood lactate response to incremental PE until fatigue is well established.While its concentration is maintained close to 1 mmol/L in conditions of low to moderate work load, in situations of greater loads there is a non-linear increase to maximum VO 2 25 .Blood lactate varied similarly in both experimental conditions, but was accentuated more in hypoxia, particularly immediately after the condition.This behavior differs from other studies highlighting the intriguing phenomenon of the lactate paradox.The lactate paradox refers to an unexpected reduction of blood lactate concentration during PE in individuals recently exposed and/or acclimatized to high altitudes 26 .
In contrast with this explanation and consistent with our results, Reeves et al. 27 argued that the accumulation of blood lactate during PE increases in higher altitudes, but reduces after acclimatization.This is paradoxical because that this occurs without any change in the delivery of oxygen to the muscle.This finding was also observed by Van Hall et al. 28 , where individuals at sea level engaged in a regimen of incremental PE until exhaustion, had increased arterial blood and muscle lactate concentrations in an acute exposure to hypoxia (simulated at 4100m) condition.
This behavior could be possibly explained by anaerobiosis, increased glycolytic flow 29 , and elevated levels of catecholamines 30 .However, despite the efforts of numerous studies, there is a lack of conclusive and convincing evidence for this phenomenon.

CONCLUSIONS
The particularities of a hypoxic environment associated with maximum PE can result in decreases in mood and physiological responses that possibly could negatively modulate physical performance.

Figure 1 .
Figure 1.Experimental design followed by 12 volunteers undergoing a TEmax regimen in two experimental conditions, sea level and hypoxia simulated at an altitude of 4,500 meters.The experimental preparation of the participants was 15 minutes, allowing participants to be placed and prepared in the chamber for the beginning of the procedure.After these adjustments, the participants commenced the TEmax.Subjective and physiological evaluations were performed at baseline, follow up (immediately after), 30 minutes, and 60 minutes after TEmax, in their respective experimental conditions.

Figure 2 .
Figure 2. Mood Profile assessed by the BRUMS instrument and their respective dimensions, observed at TEmax in both experimental conditions, at all times.T -tension/Anxiety; D -Depression; R -Anger/hostility; V -vigor; F -fatigue; CM -mental confusion.Repeated measures ANOVA with Duncan post hoc tests.Data presented as mean  standard error, with significant results for p≤0.05. a -significantly different from baseline, in the same condition; b -significantly different from follow up immediately after, in the same condition; c -significantly different from sea level condition, at the same time.

Figure 3 .
Figure 3. Oxyhemoglobin saturation observed in the TEmax, in both experimental conditions, at all times.Repeated measures ANOVA with Duncan post hoc tests.Data presented as mean ± standard deviation-pattern, with significant results for p≤0.05.-different from baseline, in the same condition; b -different from immediately after, in the same condition; c -different from 30 minutes, in the same condition; d -different from sea level condition, at the same time.

Figure 4 .
Figure 4. Concentration of blood lactate, observed in the TEmax in both experimental conditions, at all times.Repeated measures ANOVA with Duncan post hoc tests.Data presented as mean ± standard deviation-pattern, with significant results for p≤0.05.-different from baseline, in the same condition; b -different from immediately after, in the same condition; c -different from 30 minutes, in the same condition; d -different from sea level condition, at the same time.

Table 1 .
Cardiorespiratory parameters of TEmax performed at sea level.Data presented as mean ± standard deviation, referring to TEmax of 12 volunteers.VO 2 Peak -peak oxygen consumption; HR-Heart rate; VE-ventilation; VO 2 LV-I -oxygen consumption at the ventilatory threshold 1; HR LV-I -Heart rate at ventilatory threshold I; VO 2 LV-II -oxygen consumption at the ventilatory threshold 2; HR LV-II -Heart rate at ventilatory threshold II.

Table 2 .
Mood Profile on the VAMS instrument and their respective dimensions, observed at TEmax in both experimental conditions, at all times.