Sitting time is a prevalent health risk among office-based workers. To examine, using a pilot study, the efficacy of an intervention to reduce office workers' sitting time. Quasi-experimental design with intervention-group participants recruited from a single workplace that was physically separate from the workplaces of comparison-group participants. Office workers (Intervention, n=18; Comparison, n=14) aged 20–65 years from Brisbane, Australia; data were collected and analyzed in 2011. Installation of a commercially available sit–stand workstation. Changes from baseline at 1-week and 3-month follow-up in time spent sitting, standing, and stepping at the workplace and during all waking time (activPAL3 activity monitor, 7-day observation). Fasting total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, and glucose levels were assessed at baseline and 3 months (Cholestech LDX Analyzer). Acceptability was assessed with a 5-point response scale (eight items). The intervention group (relative to the comparison group) reduced sitting time at 1-week follow-up by 143 minutes/day at the workplace (95% CI= −184, −102) and 97 minutes/day during all waking time (95% CI= −144, −50). These effects were maintained at 3 months (−137 minutes/day and −78 minutes/day, respectively). Sitting was almost exclusively replaced by standing, with minimal changes to stepping time. Relative to the comparison group, the intervention group increased HDL cholesterol by an average of 0.26 mmol/L (95% CI=0.10, 0.42). Other biomarker differences were not significant. There was strong acceptability and preference for using the workstations, though some design limitations were noted. This trial is the first with objective measurement and a comparison group to demonstrate that the introduction of a sit–stand workstation can substantially reduce office workers' sitting time both at the workplace and overall throughout the week.