The purpose of this research was to compare the effect of an exhaustive exercise session on the concentration of serum testosterone and cortisol and cortisol/testosterone ratio in the morning and the evening in female volleyball players. For this purpose, 12 female volleyball players (mean age 23.26 years, mean weight 58.8 kg, mean height 166.49 cm and at least four years of activity) participated voluntarily in this research. Bruce Test was performed on a treadmill in the morning and evening. The blood samples were collected before and after of Bruce test. The levels of these hormones were measured by Eliza method. SPSS 16 software was used to statistically analyze the data; descriptive statistics were used to describe the data and to investigate the characteristics of the groups and t test was used to compare the groups. The results showed no significant difference in testosterone and testosterone/cortisol ratio between the morning and evening; but an exhaustive exercise session significantly decreased cortisol in the morning and evening. It can be concluded that an exhaustive exercise session cannot affect testosterone and testosterone/cortisol ratio while it may significantly reduce cortisol.