Prolonged periods of sitting have been linked to negative health outcomes. Implementation of sit-stand desks in the workplace has been one strategy to reduce prolonged sitting. To assess the effectiveness of sit-stand workstations on reducing sitting time and improving other health outcomes of office-based workers. 39 Portuguese office workers were randomized into a 6-month parallel-group cluster RCT consisting by the implementation of sit-stand desks in the workplace. The primary outcome of sitting time was assessed using ActivPAL. Secondary outcomes included biometric, psychological, and diet-related variables. All outcomes were assessed at baseline and 6 months for the whole sample and at 3 months for a sub-sample of the intervention group (n = 11). No significant time*group interaction was found for the primary or secondary outcomes, apart from waist circumference favoring the control group (Δ-1.81 cm, pinteraction = 0.04). There were significant changes within the intervention group for sitting time (-44.0 min/day), prolonged sitting (>30 min) (-45.3 min/day) and standing time (51.7 min/day) at 3 months in the sub-sample and in prolonged sitting (>30 min) (-26 min/day) in the full intervention group (p < 0.05). Changes were also observed within the intervention group for percent body fat (Δ-3.7%) and ratings of quality of life (Δ2.2), musculoskeletal discomfort (Δ-4.9), overall fatigue (Δ-2.2), and the need for recovery after work (Δ-1.7) at 6-month follow-up (p < 0.05). Although not being effective for reducing sitting time, the implementation of sit-stand desks in the Portuguese workspace was shown to be feasible over the long term, received well by users, and may offer other health benefits. OSF Registration, OSF.IO/JHGPW. Registered 15 November 2022. https://doi.org/10.17605/OSF.IO/JHGPW.