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The aim of this study was to evaluate the effect of an activity-based work (ABW) office environment on physical activity and sedentary behavior, work ability, and musculoskeletal discomfort.Eighty-eight office workers trialed ABW for 4 weeks. Accelerometer and self-reported outcomes were measured at baseline, end-intervention, and follow-up. Data were analyzed using linear mixed models.Accelerometry measured sedentary time; sedentary breaks and step count did not significantly change from baseline to end-intervention (P = 0.13, 0.09, 0.18, respectively). Self-reported sitting-time was 14% lower, with standing-time and walking 11% and 3% higher in ABW than baseline (P < 0.01 for all). Low back pain was lower in ABW than baseline (odds ratio 2.0, 95% confidence interval 1.1 to 3.7). Work ability reduced from 8.4 to 7.8 points (P < 0.01) at follow-up.ABW environment appears to reduce self-reported sedentary behavior and low back pain and increase standing time.