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Office environments have been causally linked to workplace-related illnesses and stress, yet little is known about how office workstation type is linked to objective metrics of physical activity and stress. We aimed to explore these associations among office workers in US federal office buildings.We conducted a wearable, sensor-based, observational study of 231 workers in four office buildings. Outcome variables included workers’ physiological stress response, physical activity and perceived stress. Relationships between office workstation type and these variables were assessed using structural equation modelling.Workers in open bench seating were more active at the office than those in private offices and cubicles (open bench seating vs private office=225.52 mG (31.83% higher on average) (95% CI 136.57 to 314.46); open bench seating vs cubicle=185.13 mG (20.16% higher on average) (95% CI 66.53 to 303.72)). Furthermore, workers in open bench seating experienced lower perceived stress at the office than those in cubicles (−0.27 (9.10% lower on average) (95% CI −0.54 to −0.02)). Finally, higher physical activity at the office was related to lower physiological stress (higher heart rate variability in the time domain) outside the office (−26.12 ms/mG (14.18% higher on average) (95% CI −40.48 to −4.16)).Office workstation type was related to enhanced physical activity and reduced physiological and perceived stress. This research highlights how office design, driven by office workstation type, could be a health-promoting factor.