The neural mechanisms underlying feelings of fatigue are poorly understood. The primary purpose of the study was to use functional magnetic resonance imaging (fMRI) to determine the association between feelings of mental fatigue and blood oxygen level dependent (BOLD) brain responses during a mentally fatiguing cognitive task. Healthy, non-fatigued controls and chronic fatigue syndrome (CFS) patients were included to determine the influence of chronic levels of fatigue on brain responses. We hypothesized that mental fatigue would be significantly related to brain activity during a fatiguing cognitive task but not during either a non-fatiguing motor (finger tapping) or cognitive (auditory monitoring) task. Patients (n=9) and controls (n=11) completed a finger tapping task, a simple auditory monitoring task and a challenging working memory task, designed to induce mental fatigue, while undergoing fMRI. Fatigue was measured prior to scanning and following each task during fMRI data collection. Results showed that mental fatigue was significantly related to brain activity during the fatiguing cognitive task but not the finger tapping or simple auditory monitoring tasks. Significant (p< or =0.005) positive relationships were found for cerebellar, temporal, cingulate and frontal regions. A significant (p=0.001) negative relationship was found for the left posterior parietal cortex. CFS participants did not differ from controls for either finger tapping or auditory monitoring tasks, but exhibited significantly greater activity in several cortical and subcortical regions during the fatiguing cognitive task. Our results suggest an association between subjective feelings of mental fatigue and brain responses during fatiguing cognition.