We sought to investigate if critical oxygenation (COx) is a robust marker of exercise intensity, and if it remains stable in normoxia and hypoxia with simultaneous changes in critical power (CP) and heart rate (HR).Thirty-three highly trained endurance athletes (11 females) underwent two 3-min CP cycling tests in normoxia (87 m ASL, FiO2 = 20.8%) and normobaric hypoxia (3200 m ASL, FiO2 = 14.2%). Repeated measures ANOVA with partial eta (ηp2) and omega squared (ω2) effect sizes was employed to compare systemic (SpO2) and muscle oxygen saturation (SmO2) at rest and COx, HR, and CP during exercise between normoxia and hypoxia with biological sex as an independent variable. Bayesian T-tests were conducted as the confirmatory analysis. Significant differences between normoxia and hypoxia for SpO2 and SmO2 were observed at rest in both sexes. During exercise, COx in the triceps brachii, CP and various HR indices exhibited significant differences (p textless 0.001), whereas differences were not significant in the vastus lateralis (p = 0.355). The Bayesian analysis supported these findings. The decrease in COx in the triceps brachii in hypoxia was larger in females than in males (30 vs. 21% drop respectively, p = 0.019). However, no environment×sex interaction was found for CP, HR, and COx in vastus lateralis. COx in locomotor muscles remains stable across the tested ambient oxygen concentrations, whereas CP and HR exhibit significant differences between normoxia and hypoxia. Accordingly, COxVL may be useful in optimizing training load and cycling performance under different oxygen availability conditions.