Subject and Parameter Selection

The detailed methodology was of the EROS study was previously published (18–22), also available at a depository (https://osf.io/bhpq9/). The present study was approved by the ethical committee of the Federal University of São Paulo (Approval Number: 1093965).

We recruited participants through social media (Facebook, Instagram, Whatsapp), and were initially evaluated for age, sex, weight and height, clinical characteristics, and if they were suspected for Overtraining Syndrome (OTS), healthy athletes (ATL), or non-physically active (NPAC), and training (if athletes). Inclusion criteria for all participants, criteria for all athletes, and specific criteria for OTS are shown in Figure 2. Employing a two-step selection process, we avoided athletes with an insufficient amount of training for the adaptive changes to exercises, non-full sedentary, extremes of age, misdiagnosis of OTS, and presence of confounding hormones, medications, and diseases.

Selection criteria for the EROS study.

For OTS candidates, we employed the diagnostic flowchart proposed by the latest guideline on OTS (15), the joint guideline of the European College of Sport Science and American College of Sports Medicine, from 2013, which requires the presence of decrease of at least 10% in training intensity, volume, pace, power, strength, or overall performance, decrease of the time-to-fatigue of at least 20%, both verified by a professional coach, increased sense of effort, changes in behavior and decreased energy levels, with or without sleep disturbances, infections or injuries, which persisted for at least 1–2 months, despite the attempts to recover, and which is not caused by conditions that could inherently lead to decrease of performance, including inflammations, infections, and frank hormonal dysfunctions.

All selected participants signed a written informed consent for participation in the study, approved by the ethics committee of the Federal University of São Paulo, in accordance with the Declaration of Helsinki. Then, participants underwent hormonal responses to stimulation tests, basal biochemical, inflammatory, muscular, immunologic, and hormonal parameters, nocturnal urinary catecholamines (NUC) and its metabolites, analysis of body metabolism and composition, and evaluation of psychological, social, sleep, and eating patterns.

In the EROS-HPA axis arm of the study, we evaluated peripheral and central components of the hypothalamic-pituitary-adrenal (HPA) axis (whether primary or peripheral: adrenal, or central: pituitary and hypothalamus), by employing a 250 ug cosyntropin stimulation test (CST), for direct evaluation of cortico-adrenal responses to a synthetic ACTH, an insulin tolerance test (ITT), an exercise-independent stimulation test that provokes a hypoglycemia as the stimulation for the evaluation of the integrity of the HPA axis, and salivary cortisol rhythm (SCR), for the identification of the patterns of the circadian rhythm of the cortisol.

In the EROS-STRESS test we employed the same ITT for the evaluation of the of the growth hormone (GH) and prolactin responses to hypoglycemia, and which we detailed different aspects of the test, including time-to-hypoglycemia, glycemic nadir, severity of adrenergic, and neuroglycopenic symptoms during hypoglycemia, and compared between groups.

In the EROS-BASAL arm, we evaluated basal hormones, immunologic, muscular, classical inflammatory, lipids, and vitamin. And for the EROS-PROFILE, participants underwent several questions regarding specific sleeping, eating, social, and psychological patterns, and underwent analysis of body composition and metabolism.

The full process was performed during a short period, of <10 days, between the recruitment, clinical, and biochemical inclusion and exclusion criteria, the collect of the basal biochemical parameters, all questionnaires, body composition, and metabolism, and functional tests. For all parameters we performed three-group and pairwise comparisons.