To evaluate the construct validity of Psychomotor Vigilance Test performance for measuring fatigue in people with acquired brain injury. Observational cross-sectional study. Fifty-four people with acquired brain injury and 61 healthy controls. Participants performed the Psychomotor Vigilance Test and reported momentary fatigue before and after this test and general fatigue. Associations between performance and fatigue in patients were tested by correlational and hierarchical multiple linear regression analyses, controlling for sleep quality, daytime sleepiness, and mood. Patients performed worse on the test compared with controls. Within the patient group, worse test performance was associated with increases in momentary post-test fatigue and general fatigue, indicating convergent validity, but also with daytime sleepiness, and mood complaints, indicating a lack of divergent validity. When controlling for sleepiness and mood, the association between performance and general fatigue was no longer significant, whereas the association between performance and post-test fatigue remained. Performance on the Psychomotor Vigilance Test cannot be used as a specific measure for fatigue, but it appears to be a more general measure of severity of symptoms including fatigue, mood, and sleepiness. Therefore, the Psychomotor Vigilance Test may be a useful measure to examine the effects of interventions aimed at reducing these symptoms.