Background. Intense physical exercise influences the secretion of hormones involved in several metabolic processes. In particular, an acute cortisol (C) and growth hormone (GH) increase after an acute physical activity is widely demonstrated in literature. Moreover, the testosterone (T)/C ratio was recently proposed as a performance index in male athletes. On the contrary, little is known about hormonal changes in women under physical activity and only few studies evaluated the hormonal trend in an extended period of time. Aim. To evaluate the hormonal changes during an annual volleyball female regular season. Methodology. A longitudinal, retrospective, observational clinical trial was carried out. 28 female professional volleyball players belonging to the same team were enrolled. For each subject, blood samples were collected after 36 hours of rest in four visits. Visit 1 was performed at the beginning of the training phase, visit 2 and 3 were performed during and Visit 4 were performed at the end of the regular season. GH, insulin-like growth factor (IGF)-1, T and C were assessed by immunoenzimatic assays. Results. Both GH and IGF-1 were above the normal range at visit 1 (5.08±5.22 ng/ml; 265.88±105.85 ng/ml). In particular, 5 (17.8%) athletes had GH above the normal range. No alterations of other hormones were found at Visit 1. C significantly changed during the regular season (p=0.009), with higher levels at Visit 2 (p=0.049), compared to visit 1 and the following visits. Similarly, T significantly changed during the season (p=0.013), even if a clear trend was not demonstrated. The post-hoc test showed two peaks of T at Visit 2 and Visit 4 (p=0.029 and p=0.024, respectively). Accordingly, the T/C ratio significantly changed during visits (p=0.009), decreasing of about 30% in the first phase of the regular season, suggesting an overreaching of subjects enrolled. Conclusion. Here we found that chronic intense physical activity influences hormonal levels in female volleyball players. In particular, C secretion is increased at the beginning of the regular season, whereas T serum levels shows a significant fluctuation during the regular season. Moreover, we suggest that T/C ratio could be useful in the evaluation of the training overload in female athletes.