Background: Burnout and prolonged fatigue are related but distinct concepts that have seldom been empirically compared. Aim: To examine similarities, overlap and differences between burnout and prolonged fatigue. Design: Observational study. Methods: We analysed baseline data from the Maastricht Cohort Study on Fatigue at Work (n = 12 140). The discriminative abilities of the Checklist Individual Strength (CIS) and the Maslach Burnout Inventory - General Survey (MBI-GS) were evaluated using principal component analysis. Overlap, similarities and differences regarding health, work and demographic factors between subgroups were assessed. Results: The discriminative abilities of the CIS and MBI-GS appeared to be moderate. Prolonged fatigue and burnout cases overlapped considerably. The subgroup consisting of cases with concurrent fatigue and burnout tended to have poorer outcomes in terms of health and work factors than the subgroups with either prolonged fatigue or burnout. Similar patterns were found for subjective fatigue and exhaustion. Discussion: There appear to be some relevant differences between burnout and prolonged fatigue, with respect to work and health factors. Burnout and prolonged fatigue can occur both separately and simultaneously. Having both conditions simultaneously seems to be associated with worse outcomes than having either alone.