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Objective This study examined office workstation types' impact on the relationship between fatigue and three health metrics: physical activity, stress, and sleep quality. Methods Data from 225 office workers were collected for perceived fatigue, perceived sleep quality (Pittsburgh Sleep Quality Index [PSQI]), physiological stress response (standard deviation of heart rate variability [HRV]), and physical activity (total activity in minutes) during three consecutive workdays. Stress and physical activity were measured using chest-worn sensors. Workers were then categorized as tired or not-tired based on the median of the fatigue rating. Results Among tired workers, open-bench seating workers had increased physical activity, improved sleep quality, and reduced stress compared with workers in private offices and cubicles. Conclusions Office workstation types influence physical activity and levels of stress during work hours, which in turn affect sleep quality.