Purpose <p>Workplace health interventions with exercise have positive effects on musculoskeletal pain and well-being at work, however, effectiveness is questioned due to low adherence. In hospitals participation is challenged by shiftwork and unpredictable workload. Our aim was to investigate the feasibility of exercise during working hours in an acute hospital, herein to estimate the health impact to guide management decisions on implementation.</p> Methods <p>A clinical trial in a public hospital, offering staff supervised group-based individualized exercise with combined aerobic and strength training during working hours twice weekly for 20&#xa0;weeks. Delivery, acceptance, and adherence were investigated. Subjective outcomes were social capital, well-being, quality of life, and musculoskeletal pain were assessed. Objective outcomes were blood pressure, body composition, and cardiorespiratory fitness.</p> Results <p>Twenty-three percent of the employees (<i>n</i> = 617) accepted participation (92% female, median age was 50&#xa0;years, 38% nurses). Adherence was 29% with no difference between employees with clinical versus non-clinical functions<i>.</i> Non-clinicians participated during working hours, while clinicians participated outside of working hours in 50% (IQR 5–87) of the sessions. Positive changes were seen in systolic and diastolic blood pressure (decreased 2.0 [0.9; 2.2] and 0.9 [0.1; 1.7] mmHG, respectively), aerobic capacity 2.3&#xa0;ml/O2/min/kg [1.7; 2.9], and in waist-hip ratio, social capital, well-being, quality of life, and musculoskeletal pain.</p> Conclusion <p>Exercise during working hours in an acute hospital staff was feasible, but strategies to increase acceptance and adherence are necessary for a successful implementation. Despite low adherence, the intervention was associated with improvements of physical and mental health. Registration: The study protocol has been uploaded on <a href="http://www.clinicaltrials.gov">www.clinicaltrials.gov</a> (NCT04988724).</p>