Research examining the prevalence, physical activity (PA) and sedentary behaviour (SB) in shift workers show mixed results. This systematic review and meta-analysis aimed to compare PA and SB in shift workers with non-shift workers following the PRISMA guidelines. Ebscohost megafile ultimate (CINHAL, E-journals, Academic search ultimate, health source consumer edition, SPORT Discus), PubMed, Scopus, Web of Science, and Science Direct databases were searched up to April 2021. Cross-sectional and baseline data from longitudinal studies reporting PA and SB in full time workers were eligible. Data on participants characteristics and time spent in PA and SB and/or prevalence of workers meeting PA guidelines were extracted and pooled with random effects model. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) 10-item checklist was adapted and used. A total of 49 studies met inclusion criteria and 21 studies included for meta-analysis. The prevalence of meeting physical activity guidelines (OR 0.84, 95% CI: 0.68, 1.03) and standardized mean difference (SMD) of time spent in moderate-to-vigorous physical activity (SMD −0.1, 95% CI: −0.4, 0.20) were similar in shift and non-shift workers. Time spent in sedentary behaviour was lower in shift workers than non-shift workers (SMD −0.2, 95% CI: 0.50, −0.001). While the differences in PA are not so evident between shift and non-shift workers, the prevalence of sufficient PA was low in both groups. These preliminary findings provide support for inclusion of physical activity and sedentary behaviour in health promotion interventions targeted at shift workers.