The influence of menstrual cycle phase and fitness status on metabolism during high-intensity interval exercise (HIIE) was assessed. Twenty-five females (24.4(3.6) yr) were categorized by normal menstrual cycle (n = 14) vs. oral contraceptive use (n = 11) and by aerobic fitness, high fit females (HFF: n = 13) vs. low fit females (LFF: n = 12). HIIE was four sets of four repetitions with 3 min rest between intervals on a cycle ergometer at a power output halfway between the ventilatory threshold and V̇O2peak and performed during follicular (FOL: day 2-7 or inactive pills) and luteal phases (LUT: day ~21 or 3rd week of active pills). Substrate oxidation was assessed via indirect calorimetry, blood lactate via finger stick, and recovery skeletal muscle oxidative metabolism (mV̇O2) via continuous-wave near-infrared spectroscopy. HFF oxidized more fat (g•kg-1) during the full session (FOL: p = 0.050, LUT: p = 0.001), high intervals (FOL: p = 0.048, LUT: p = 0.001), low intervals (FOL: p = 0.032, LUT: p = 0.024) and LUT recovery (p = 0.033). Carbohydrate oxidation area under the curve was greater in HFF during FOL (FOL: p = 0.049, LUT: p = 0.124). Blood lactate was lower in LFF in FOL (p ≤ 0.05) but not LUT. Metabolic flexibility (Δ fat oxidation g•kg-1•min-1) was greater in HFF than LFF during intervals 2-3 in FOL and 1-4 in LUT (p ≤ 0.05). Fitness status more positively influences exercise metabolic flexibility during HIIE than cycle phase or OC use.