PurposeTo assess the transferability of the reference intervals (RI) of testosterone (T), cortisol (C), human growth hormone (hGH), and insulin‐like growth factor (IGF)‐1, calculated on a normal healthy population, to a population of female elite volleyball players. Secondary aim of this study is the evaluation of the T/C ratio as predictive tool of overtraining during the annual regular season.MethodsA retrospective, longitudinal, observational study was performed, enrolling 58 professional female volleyball players periodically evaluated during the regular sportive season, which lasts from September to May.ResultsStatistically significant differences between the volleyball players and reference populations for T (P = .010), C (P < .001), and IGF‐1 (P < .001) were found. Three different statistical approaches to calculate the RI in the athlete group showed a high degree of concordance and pointed out a shift upwards of both lower and upper reference limits. The T/C ratio significantly changed among visits (P = .009). In particular, an overall decrease of about 30% was observed for this ratio during the season, suggesting a state of overtraining.ConclusionT, C, hGH, and IGF‐1 reference values calculated on elite volleyball female players are higher than those of the reference population used in normal clinical practice, suggesting that the health status of highly trained subjects needs the definition of tailored RI for these variables. Moreover, the utility of T/C ratio in the evaluation of overtraining is confirmed.