Ovarian hormones may modulate key physiological functions that play a crucial role in the acute response to hypoxia. Women remain underrepresented in high-altitude physiology research. This exploratory study aimed to investigate the impact of menstrual cycle (MC) phases on resting skeletal muscle oxygen consumption and post-occlusive microvascular reactive hyperemia in the lower limbs during acute high-altitude exposure in eumenorrheic women. Microvascular function was assessed via vascular occlusion test in combination with near-infrared spectroscopy on the vastus lateralis muscle. Measurements were conducted at low altitude (1224 m) and after one night at 3375 m (inspired O2 pressure: 96 ± 1 mmHg) during both the early follicular (EF) and mid-luteal (ML) phases. At high altitude, baseline tissue saturation index (TSI) (65.0 ± 4.8 vs. 66.1 ± 2.7 %; p = 0.559), desaturation rate (−0.086 ± 0.061 vs. −0.080 ± 0.039 %·s−1; p = 0.920), normalized reperfusion slope (0.013 ± 0.010 vs. 0.014 ± 0.005 %·s−1; p = 0.100) and minimum TSI (52.9 ± 6.8 vs. 53.9 ± 3.9 %; p = 0.647) did not differ significantly between EF and ML. Reperfusion rate decreased significantly from low (0.894 ± 0.320) to high altitude during both EF (0.661 ± 0.424; p = 0.027) and ML (0.722 ± 0.253; p = 0.027). These findings suggest that microvascular function is not significantly modulated by the MC at 3375 m. This study adds further evidence suggesting that no specific recommendation regarding the optimal menstrual cycle phase for acute high-altitude exposure is warranted.