Mental fatigue is a psychobiological state caused by prolonged periods of demanding cognitive activity. Although the impact of mental fatigue on cognitive and skilled performance is well known, its effect on physical performance has not been thoroughly investigated. In this randomised cross-over study, 16 subjects cycled to exhaustion at 80% of their peak power output after 90 min of a demanding cognitive task (mental fatigue) or 90 min of watching emotionally neutral documentaries (control). After experimental treatment, a mood questionnaire revealed a state of mental fatigue (P = 0.005) which significantly reduced time to exhaustion (640 ± 316 s) compared to the control condition (754 ± 339 s) (P = 0.003). This negative effect was not mediated by cardio-respiratory and musculo-energetic factors as physiological responses to intense exercise remained largely unaffected. Self-reported success and intrinsic motivation related to the physical task were also unaffected by prior cognitive activity. However, mentally fatigued subjects rated perception of effort during exercise to be significantly higher compared to the control condition (P = 0.007). As ratings of perceived exertion increased similarly over time in both conditions (P < 0.001), mentally fatigued subjects reached their maximal level of perceived exertion and disengaged from the physical task earlier than in the control condition. In conclusion, our study provides experimental evidence that mental fatigue limits exercise tolerance in humans through higher perception of effort rather than cardio-respiratory and musculo-energetic mechanisms. Future research in this area should investigate the common neurocognitive resources shared by physical and mental activity.