Research into the effects of exercise on the modulation of testosterone levels in males and females has been performed in both clinical and preclinical studies. These studies have demonstrated that males tend to experience an increase in serum testosterone levels from exercise, while females do not. However, few studies have directly compared the modulation of testosterone levels between males and females under identical chronic exercise regimens and conditions. This study divides male and female rats into four separate groups to try to determine the effect a chronic treadmill exercise regimen (six weeks) on testosterone modulation, as well as the sex differences present in this modulation. The male and female rats were each equally divided into sedentary and exercise groups. In this study, blood samples were collected from male and female rats from each of the four groups. These samples were used to quantify testosterone levels using an enzyme-linked immunosorbent assay (ELISA) plate immunoassay. The results showed that the exercised male rats had significantly greater testosterone levels (137.2% increase) than the sedentary males. These data support the notion that chronic treadmill exercise can induce an increase in testosterone levels in male rats. In contrast, female rats did show an increase in testosterone levels (76.14%), but it was not statistically significant compared to the sedentary group. These results support the hypothesis that chronic treadmill exercise can induce an increase in serum testosterone levels and that this is a sex-specific effect. Further research is needed into the relationship between exercise and testosterone levels.