Objective: The goal of this review was to summarize the evidence for an effect of lifestyle-targeted interventions at the workplace on the main biological risk factors for cardiovascular disease (CVD). Methods: We performed an extensive systematic literature search for randomized controlled trials (RCT) that met the following inclusion criteria: (i) targeted at workers; (ii) aimed at increasing physical activity and/or improving diet; and (iii) measured body weight, body fat, blood pressure, blood lipids and/or blood glucose. We used a nine-item methodological quality list to determine the quality of each study. A best-evidence system was applied, taking into account study quality and consistency of effects. Results: Our review included 31 RCT, describing a diversity of interventions (eg counseling, group education, or exercise). Of these studies, 18 were of high quality. Strong evidence was found for a positive effect on body fat, one of the strongest predictors of CVD risk. Among populations "at risk", there was strong evidence for a positive effect on body weight. Due to inconsistencies in results between studies, there was no evidence for the effectiveness of interventions on the remaining outcomes. Conclusions: We found strong evidence for the effectiveness of workplace lifestyle-based interventions on body fat and, in populations at risk for CVD, body weight. Populations with an elevated risk of CVD seemed to benefit most from lifestyle interventions; supervised exercise interventions appeared the least effective intervention strategy. To gain better insight into the mechanisms that led to the intervention effects, the participants' compliance with the intervention and the lifestyle changes achieved should be reported in future studies. This work is licensed under a Creative Commons Attribution 4.0 International License.