Acute Effects of Different Training Sessions on the Extended Blood Steroid Profile in Elite Male Football Players

Background: Acute exercise is a recognized physiological confounder of circulating steroid hormones, yet evidence on how real-world football training affects the blood steroid profile (BSP) within the Athlete Biological Passport (ABP) remains limited in elite athletes. Objective: To examine acute endocrine response to football training sessions of differing load and to explore their association with perceived exertion. Methods: A total of 27 elite male professional outfield football players from an Italian Serie A club were assessed during the in-season period (2022-2023 competitive season). Each player completed one predefined low-load (LL), moderate-load (ML), or high-load (HL) training session. Venous blood samples were collected immediately before and after training. Collectively, 26 circulating steroids were quantified by liquid chromatography-tandem mass spectrometry. These included both unconjugated hormones and phase II metabolites relevant to the BSP. External training load was assessed using global navigation satellite systems, while internal load was quantified via heart-rate telemetry and rating of perceived exertion (RPE). Data were analyzed using nonparametric statistics, linear mixed-effects models, and multivariable logistic regression. Results: Training elicited significant, load-associated alterations in circulating BSP steroids. HL sessions induced marked post-training increases in free glucocorticoids including cortisone (median Δ + 7.4 ng/mL), and unconjugated androgens such as androstenedione (median Δ + 131.3 pg/mL), whereas LL sessions induced minimal changes. The testosterone-to-androstenedione ratio decreased following ML and HL training (median 8.2-6.6, p < 0.001). Athletes reporting higher perceived exertion (RPE ≥ 4) exhibited greater endocrine perturbations, with significantly larger increases in cortisol-related steroids (e.g., 11-deoxycortisol, p < 0.001). Conclusions: Acute football training is associated with rapid modulation of circulating steroid hormones, particularly following higher training loads and perceived exertion. These findings highlight the potential relevance of recent training exposure when interpreting BSP variations and suggest that integrating steroidomic profiles with training-load information may improve the physiological contextualization of antidoping biomarkers.