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Objective: To investigate the effects of sustained mental activity on perceptions of mental fatigue, cognitive performance, and autonomic response in patients with clinical burnout as compared to a healthy control group. Methods: Patients with clinical burnout (n = 30) and healthy control participants (n = 30) completed a 3-hour test session, in which they were administered a set of cognitive tests before and after an effortful cognitive task with concurrent sound exposure. Perceptions of mental fatigue and task demands (mental effort and concentration difficulties) were assessed repeatedly over the course of the test session. Heart rate variability was recorded to index autonomic response. Results: In comparison with controls, perceived mental fatigue increased earlier in the session for the clinical burnout group and did not recover following a short rest period. Throughout the session, patients rated the tasks as more demanding and showed less improvement on measures of attention and processing speed, inhibition and working memory. While autonomic responses were initially comparable, there was a unique decrease in high-frequency heart rate variability in the clinical burnout group after extended testing and exposure. Conclusion: Patients with clinical burnout are affected differently than healthy controls by sustained mental activity, as reflected by ratings of perceived mental fatigue, aspects of cognitive performance and autonomic response. Further investigation into the role of autonomic regulation in relation to cognitive symptoms in clinical burnout is warranted.