Background Replacing sitting with standing is one of several recommendations to decrease sedentary time and increase the daily energy expenditure, but the difference in energy expenditure between standing versus sitting has been controversial. This systematic review and meta-analysis aimed to determine this difference. Designs and methods We searched Ovid MEDLINE, Ovid Embase Scopus, Web of Science and Google Scholar for observational and experimental studies that compared the energy expenditure of standing versus sitting. We calculated mean differences and 95% confidence intervals using a random effects model. We conducted different predefined subgroup analyses based on characteristics of participants and study design. Results We identified 658 studies and included 46 studies with 1184 participants for the final analysis. The mean difference in energy expenditure between sitting and standing was 0.15kcal/min (95% confidence interval (CI) 0.12-0.17). The difference among women was 0.1kcal/min (95% CI 0.0-0.21), and was 0.19kcal/min (95% CI 0.05-0.33) in men. Observational studies had a lower difference in energy expenditure (0.11kcal/min, 95% CI 0.08-0.14) compared to randomised trials (0.2kcal/min, 95% CI 0.12-0.28). By substituting sitting with standing for 6 hours/day, a 65kg person will expend an additional 54kcal/day. Assuming no increase in energy intake, this difference in energy expenditure would be translated into the energy content of about 2.5kg of body fat mass in 1 year. Conclusions The substitution of sitting with standing could be a potential solution for a sedentary lifestyle to prevent weight gain in the long term. Future studies should aim to assess the effectiveness and feasibility of this strategy.