Before discussing the hormonal changes themselves, it is important to note that the daily variation in weight during the walk was small, and this minimal change is a consequence of the frequent supply of fluids and food throughout the entire walk, which prevented dehydration and caloric deficits that could interfere with hormonal assessments [
5,
9]. An additional aspect that had some influence in our findings is the fact that all participants were well trained amateur athletes, who most certainly had an attenuated hormonal response to exercise when compared to untrained individuals [
10].
Cortisol
Analysis of the variation in cortisol showed a gradual decrease in blood levels throughout the days of the walk, with a significant decreasing trend between the 1st and 4th days. This trend suggests an adaptive process to stress or may indicate that the fatigue induced by the previous exercise may have modified the hormonal response, leading to feedback suppression [
11]. According to Kraemer and Rogol [
12], a progressive adaptation occurs, with a decreased adrenal response to the adrenocorticotropic hormone (ACTH) released by exercise at the same relative intensity.
In this group of race walkers, the cortisol level measured at the end of the 1st day of walking, collected at night, was surprisingly larger than the baseline value collected in the morning, contrary to expectations based on the circadian rhythm [
13,
14]. This increase in cortisol is a consequence of the type of walking exercise, which is considered as medium to high intensity, and the prolonged duration.
The hypothalamic–pituitary–adrenal (HPA) axis is activated during stress. Corticotrophin-releasing hormone (CRH), which has a predominant role, and arginine vasopressin (AVP) play an important role in exercise-induced stimulation of ACTH secretion, which consequently leads to an increase in adrenal cortisol levels [
9,
12].
Cortisol is therefore an exercise-responsive hormone that shows a significant increase after high intensity and short duration exercise or submaximal intensity exercise with a longer duration [
15]. Therefore, the decrease trend in cortisol response found was not as expected. Although it has been reported, in a study that evaluated cortisol responses to daily strenuous walking during 4 successive days [
11], suggesting a progressive adaptive response to stress that could eventually lead to decreasing cortisol values. Since cortisol is a potential biomarker of a catabolic state [
16] our findings can be interpreted as beneficial and a chronic effect of this exercise modality.
Thus, two factors modulate the response of the HPA axis to exercise: intensity and duration [
9]. The minimum exercise intensity required to produce a cortisol response is 60% of the VO
2Max. Exercise above this threshold increases the plasma cortisol concentration linearly with exercise intensity [
9].
The activation of the HPA axis represents a physiological response to the energetic, metabolic, vascular, neurophysiological, and psychological requirements of exercise [
9]. Glucocorticoids have many beneficial effects during physical activity, including increasing the viability of metabolic substrates for the energy needs of the muscle, maintaining normal vascular integrity and responsiveness during exercise, and preventing overactivity of the immune system due to repeated, exercise-induced muscle injury [
9].
Independent of thermal stress, hypohydration potentially amplifies the physical activity-induced cortisol response through additional stimulation of AVP and ACTH secretion in a cascade [
9]. In this study, this additive increase in cortisol secretion due to dehydration was not relevant because large variations in weight, which would indicate significant losses in body water, were not observed.
Thyroid hormones
Changes in thyroid hormone levels in response to exercise, in general, are small and within the normal physiological range [
17]. The literature shows divergent results regarding the behavior of thyroid hormones in response to exercise [
5,
18‐
20], with reports of an increase, decrease, or no change in the levels of thyroid hormones, regardless of the type of exercise, intensity, and duration. These ambiguous findings are attributed to numerous confounding factors, such as nutritional status and variations in body composition [
21,
22].
Interestingly, when the thyroid function was analyzed, the curve of the FT4 levels was similar to that of cortisol: an initial increase on the 1st day, relative to the baseline value, and a subsequent significant decreasing trend (Fig.
3), as if an adaptive reaction of the thyroid occurred in response to the initial stress. Thus, exercise possibly induces an unknown signal to conserve energy that, in a cascade, would lead to decreased secretion of thyroid hormones, unrelated to changes in body composition or degree of hydration [
18]. One of the probable mechanisms would be a decrease in leptin levels, which are also affected by decreasing cortisol values, affecting specific receptors in the hypothalamus region that controls TRH expression, with consequent reduced stimulation of thyroid hormone production.
The FT4/T3 ratio showed a significant decreasing trend when the 1st and 4th days of the walk were compared (Fig.
4).
The behavior of the FT4/T3 ratio was opposite that of the metabolism of thyroid hormones during fasting and in other stress situations, i.e., reverse T3 (rT3) increases and T3 decreases as a result of the decrease in 5′-monodeiodination, generating an increase in the FT4/T3 ratio [
23,
24]. This protective physiological mechanism, which inhibits the exhaustion of the body’s energy reserves and enables the synthesis of new energy sources [
17], was likely not triggered because the food supply was practically continuous and ad libitum during the walk, thus preventing an energy deficit.
Testosterone
The effect of physical exercise on testosterone levels is also variable in the literature, with contradictory results [
4,
25,
26]. There are reports of reduction, increase, and even no change in testosterone concentrations after physical activity. These divergent results may be a consequence of differences in individual physical condition and exercise duration/intensity [
25]. In this study, a decrease in testosterone levels greater than 52% was observed relative to the baseline values collected in the morning, which is due in part to the circadian rhythm [
27] but also to the effect of exhaustive and long-duration exercise, exceeding 8 h. The slight increase on the last day of the walk can also be attributed to an adaptive effect of the axis to the effort expended.
The cortisol/testosterone ratio decreased significantly from the first to the 2nd day of the walk, remaining at these lower levels on the following days. This pattern represents a greater decrease in cortisol levels than in testosterone concentrations, with the latter remaining at more stable levels with small variations. This fact indicates a more anabolic hormonal environment, which is opposite of the effect observed for more prolonged activities [
4]. Again, the constant supply of food during the walk likely reduced the need for amino acid recruitment for gluconeogenesis, favoring a less catabolic profile [
4].
The exclusion of women in the analysis of testosterone levels is due to evidence that the levels of this androgen are very low in females, which is different from the characteristic levels of males, thus generating less reliability in the values measured [
28].
It is important to note that the only patient with diabetes showed a significant increase in blood glucose levels at the end of the 1st day of the walk but returned to lower values, considered normal, without adjustment of the dose of the hypoglycemic medication. This finding demonstrates the hyperglycemic effect of stress, largely influenced by an increase in cortisol levels, which was neutralized by the reduction of the levels of this glucocorticoid observed during the walk through the adaptive mechanism already mentioned.
One limitation of this study is that blood was collected exclusively at night (except for the baseline collection), when several hormones exhibit reduced levels due to the circadian rhythm, especially cortisol and testosterone. Other limitations related to the study design such as the fact that it was and observational study conducted without control group need to be acknowledged. Additionally, small number of individuals assessed and underrepresentation of female sex are some sample related aspects that can also be considered limitations.
However, a unique and positive feature of this study was that it was performed outside the laboratory, in the real world, without any control of environmental conditions and with high variations in temperature and humidity, people from the community, amateur athletes with a mean age greater than 40 years, and sequential assessment of a hormonal panel of great importance for the practice of physical activity.
The attenuated hormonal response to long-term moderate-to-high intensity exercise found indicate that this kind of activity is safe and harmless. The catabolic effects were lower than expected, as long as adequate nutritional and fluid replacement is provided during the physical activity.