PURPOSE: Substrate metabolism during exercise may vary across the menstrual cycle (MC) phases, likely due to estradiol (E2) and progesterone (P4). This study examined substrate metabolism during exercise in naturally menstruating (NoOC, n = 34) and women using combined oral contraceptives (COC, n = 19). METHODS: Participants were measured in a fasted state in the follicular (FOL) and luteal (LUT) phases, or the inactive (INACT) and active (ACT) phases of COC use. Serum E2 and P4 were assessed using immunoassays and body composition via bioimpedance. Peak fat oxidation (PFO) and FATMAX, the intensity eliciting PFO, were evaluated using indirect calorimetry. FATMAX was calculated using peak oxygen uptake (V̇O2PEAK), measured on the following day. RESULTS: PFO did not differ between FOL and LUT (0.40 ± 0.09 g·min-1 vs. 0.41 ± 0.10 g·min-1, p = 0.482) or INACT and ACT (0.48 ± 0.12 g·min-1 vs. 0.44 ± 0.11 g·min-1, p = 0.099). FATMAX showed no phase-related variation (NoOC: FOL 47.3 ± 15.7 % vs. LUT 47.7 ± 13.6 %, p = 0.727
COC: INACT 57.1 ± 12.3 % vs. ACT 52.5 ± 12.2 % p = 0.172). PFO was 0.08 g·min-1 (95 % confidence interval: 0.02 g·min-1-0.14 g·min-1, p = 0.010) and FATMAX 9.8 % (95 % CI: 1.0-8.7 %, p = 0.031) higher in the INACT vs. FOL. The difference in PFO persisted after adjusting for fat-free mass and V̇O2PEAK (p = 0.033) but was not significant after excluding an outlier from the COC group (p = 0.108). CONCLUSIONS: PFO and FATMAX remained stable between MC and COC phases, suggesting no need to standardize measurements by cycle phase. However, higher PFO and FATMAX in the COC group during INACT compared to FOL suggests distinct effects of exogenous hormones on metabolism compared to endogenous hormones. Practitioners should consider these differences when assessing factors influencing substrate metabolism.