Saliva represents a low stress, not-invasively collected matrix that allows steroid hormone monitoring in athletes by reflecting type, intensity and duration of exercise. Whole body cryotherapy (WBC) consists of short whole-body exposures to extremely cold air (-110° to -140°C) which, despite being initially used to treat inflammatory diseases, is currently acquiring increasing popularity in sports medicine. Cryostimulation practice is now widely accepted as an effective treatment to accelerate muscle recovery in rugby players. The aim of this work was to study the changes of steroid hormones in saliva of rugby players after both 2 and 14 consecutive WBC sessions, in order to investigate the effects of the treatment on their salivary steroid hormonal profile. Twenty-five professional rugby players, belonging to the Italian National Team, underwent a 7-day cryotherapy protocol consisting of 2 daily sessions. Saliva samples were taken in the morning prior to the start of the WBC, in the evening after the end of the second WBC, and in the morning of the day after the last WBC session. The samples were analyzed for cortisol, DHEA, testosterone and estradiol using competitive enzyme-linked immunosorbent assays. Cortisol and DHEA showed a reduction already after the 2 WBC sessions of the first day; after 14 consecutive WBC sessions cortisol, DHEA, and estradiol levels decreased, while testosterone increased as did the testosterone to cortisol ratio. These results were confirmed by the fact that the majority of subjects showed variations exceeding the critical difference (CD). In conclusion, we found that WBC acutely affects the salivary steroid hormone profile, and the results are evident already after only one twice-daily session. Most significantly, after one-week of consecutive twice-daily WBC sessions, all the hormones were modified. This is the first experimental report that links changes in the hormonal asset to WBC.