Previous pharmacological and pathological studies have reported negative relationships between circulating testosterone and certain stress hormones (i.e., cortisol and prolactin) in humans. These relationships have subsequently been used in hypotheses explaining the subclinical resting testosterone levels often found in some endurance-trained males, but as of yet no one has specifically examined these relationships as they relate to exercise. Thus, we examined the relationship between total and free testosterone levels and cortisol, and between total and free testosterone and prolactin following prolonged endurance exercise in trained males. Twenty-two endurance-trained males volunteered to run at 100% of their ventilatory threshold (VT) on a treadmill until volitional fatigue. Blood samples were taken at pre-exercise baseline (B0); volitional fatigue (F0); 30 min (F30), 60 min (F60), and 90 min (F90) into recovery; and at 24 h post-baseline (P24 h). At F0 [mean running time = 84.8 (3.8) min], exercise induced significant changes (P<0.05) from B0 in total testosterone, cortisol and prolactin. All three of these hormones were still significantly elevated at F30; but at F60 only cortisol and prolactin were greater than their respective B0 values. Free testosterone displayed no significant changes from B0 at F0, F30, or the F60 time point. At F90, neither cortisol nor prolactin was significantly different from their B0 values, but total and free testosterone were reduced significantly from B0. Cortisol, total testosterone and free testosterone at P24 h were significantly lower than their respective B0 levels. Negative relationships existed between peak cortisol response (at time F30) versus total testosterone (at F90, r=−0.53, P<0.05; and at P24 h, r=−0.60, P<0.01). There were no significant relationships between prolactin and total or free testosterone. In conclusion, the present findings give credence to the hypothesis suggesting a linkage between the low resting testosterone found in endurance-trained runners and stress hormones, with respect to cortisol.