Mental fatigue (MF) and hypoxia impair cognitive performance through changes in brain hemodynamics. We want to elucidate the role of prefrontal cortex (PFC)-oxygenation in MF. Twelve participants (22.9 ± 3.5 years) completed four experimental trials, (1) MF in (normobaric) hypoxia (MF_HYP) (3.800 m; 13.5%O2), (2) MF in normoxia (MF_NOR) (98 m; 21.0%O2), (3) Control task in HYP (CON_HYP), (4) Control in NOR (CON_NOR). Participants performed a 2-back task, Digit Symbol Substitution test and Psychomotor Vigilance task before and after a 60-min Stroop task or an emotionally neutral documentary. Brain oxygenation was measured through functional Near Infrared Spectroscopy. Subjective feelings of MF and physiological measures (heart rate, oxygen saturation, blood glucose and hemoglobin) were recorded. The Stroop task resulted in increased subjective feelings of MF compared to watching the documentary. 2-back accuracy was lower post task compared to pre task in MF_NOR and CON_NOR, while no differences were found in the other cognitive tasks. The fraction of inspired oxygen did not impact feelings of MF. Although performing the Stroop resulted in higher subjective feelings of MF, hypoxia had no effect on the severity of self-reported MF. Additionally, this study could not provide evidence for a role of oxygenation of the PFC in the build-up of MF.