Estradiol (E(2)) has been documented to have anti-inflammatory effects on the immune system. Interleukin-6 (IL-6), is classified as a "myokine", and has known metabolic consequences. Thus, the purpose of this study was to determine the effects of menstrual phase and exercise on the interaction of E(2) and IL-6, and the role of IL-6 in substrate metabolism. Ten female subjects completed three separate testing sessions: baseline evaluation, and 1 h of treadmill exercise at 65% of peak [Formula: see text] during both the midfollicular (MF) and midluteal (ML) menstrual phases. Saliva was collected prior to, during, and post-exercise for determination of E(2) and IL-6. Expired gases and an additional saliva sample were collected 30 min post-exercise. No significant differences were observed in any of the measured variables across menstrual phase. Exercise resulted in an acute rise in estradiol and IL-6; however, E(2) was not related to IL-6 at baseline or in response to exercise. IL-6 remained elevated at the end of exercise and was found to be related to energy expenditure from fat, and to total energy expenditure at 60 min, and 30 min post-exercise. No relationships were found between the anti-inflammatory estrogen E(2) and the cytokine IL-6. However, relationships were found between IL-6 and indices of substrate metabolism. Based on the data from the current research, IL-6 likely plays a metabolic role in healthy individuals during exercise when released from the muscle as a result of reduced energy availability, acting as a "myokine", in comparison to inflammation-induced IL-6 release.