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Sit–stand workstations offer a potential strategy to reduce prolonged occupational sitting. This controlled intervention study examined the effects of an environmental intervention on occupational sedentary time, musculoskeletal comfort and work ability, and the usability of sit–stand workstations in office work via a self-reported questionnaire. The intervention group (n = 24) used sit–stand workstations during the 6-month intervention period, and the control group (n = 21) used traditional sitting workstations. The results showed that working at sit–stand workstations can reduce sitting time significantly compared to control workstations (−6.7% vs. 5.0%,p = .019), which is reallocated mostly to standing (r = −0.719,p < .001). Sit–stand workstations improved perceived musculoskeletal comfort in the neck and shoulders (p = .028), as well as work ability (p = .022). The majority of intervention subjects rated sit–stand workstation adjustability as good (83.3%), and 75.0% were satisfied with the workstation. About 41.7% of the intervention participants, who were exclusively female, used the sit–stand function on a daily basis. While the environmental change alone was effective, it is likely that promoting the daily use of sit–stand workstations with counselling would lead to even more substantial positive effects. [ABSTRACT FROM PUBLISHER]