Research has shown that total testosterone (tT) levels in women increase acutely during a prolonged bout of aerobic exercise. Few studies, however, have considered the impact of the menstrual cycle phase on this response or have looked at the biologically active free testosterone (fT) form responses. Therefore, this study examined the fT concentration response independently and as a percentage (fT%) of tT to prolonged aerobic exercise during phases of the menstrual cycle with low estrogen-progesterone (L-EP; i.e., follicular phase) and high estrogen-progesterone (H-EP; i.e., luteal phase). Ten healthy, recreationally trained, eumennorrheic women (X ± SD: age = 20 ± 2 y, mass = 58.7 ± 8.3 kg, body fat = 22.3 ± 4.9 %, VO2max = 50.7 ± 9.0 ml/kg/min) participated in a laboratory based study and completed a 60-minute treadmill run during the L-EP and H-EP menstrual phases at ~70% of VO2max. Blood was drawn prior to (PRE), immediately after (POST) and following 30 minutes of recovery (30POST) with each 60-minute run. During H-EP, there was a significant increase in fT concentrations from PRE to POST (p < 0.01) while in L-EP fT levels were unchanged; which resulted in fT being significantly higher at H-EP POST versus L-EP POST (p < 0.03). Area-under-the-curve (AUC) responses were calculated, for fT the total AUC was greater in H-EP than L-EP (p < 0.04). There was no significant interaction of fT% between phases and exercise sampling time. There was, however, a main effect for exercise where fT% POST was a greater proportion of tT than at PRE (p < 0.01). In summary, hormonal changes associated with the menstrual cycle impact fT response to a prolonged aerobic exercise bout; specifically, there being higher levels under H-EP conditions. This suggests more biologically active T is available during exercise in this phase. This response may be a function of the higher core temperatures found with H-EP causing greater sex hormone binding protein release of T, or could be a function of greater degrees of glandular production. Further work is warranted to elucidate the mechanism of this occurrence. It is recommended that researchers examining T responses to exercise in women look at both tT and fT forms in order to have an accurate endocrine assessment in women.