Objective: Prolonged sitting, which is highly prevalent in office workers, has been associated with several health risks. The aim of this study was to evaluate the Dynamic Work intervention by determining its effect on total sitting time at the 8-month follow-up in comparison to the control. Methods: This two-arm pragmatic cluster randomised controlled trial included 244 office workers from 14 different departments of a large, Dutch insurance company. The Dynamic Work intervention was a real-life, worksite intervention that included environmental components (i.e. sit-stand workstations), organisational components (i.e. group sessions), and individual components (e.g. activity/sitting trackers). Outcomes were assessed at baseline, 4-month follow-up, and 8-month follow-up. The primary outcome was total sitting time per day, objectively assessed using the activPAL activity monitor at 8-month follow-up. Secondary outcomes included other total and occupational movement behaviour outcomes, health-related outcomes, and work-related outcomes. Data analyses were performed using linear and logistic mixed models. Results: Total sitting time did not differ between the intervention and control group at the 8-month follow-up. Secondary outcomes also showed no difference between the intervention and control group at either the 4-month or at 8-month follow-up, with the exception of number of occupational steps, which showed a statistically significant effect at 4-month follow-up (but not at 8-month follow-up) of 913 (95% CI = 381–1445) steps/8-h working day. Conclusions: This study evaluated the effectiveness of a real-life worksite intervention to reduce sitting time and showed little to no effect. This may be due to the relatively low intensity of the intervention, i.e. that it only involved the replacement of 25% of sitting workstations with sit-stand workstations. Future research should focus on the evaluation of more intensive real-life worksite interventions that are still feasible for implementation in daily practice. Clinicaltrials.gov, registration number: NCT03115645.