Workplace physical activity interventions in office-based settings are considered essential components of occupational health management, aimed at reducing sedentary behavior and its associated adverse effects, while also promoting employees’ health and wellbeing. This study investigates the effectiveness of office-yoga and walking on employees’ prevalence and intensity of musculoskeletal complaints (MSC), as well as on activation, which encompasses vitality and vigilance. Using a three-month longitudinal randomized controlled trial design, 459 office workers from five organizations were randomly assigned to either the office-yoga intervention group, the walking intervention group, or to a waitlist control group. Participants in the intervention groups were instructed to individually engage in brief daily sessions of the prescribed activity at the workplace. Those in the office-yoga group received video tutorials to perform the exercises. Self-reported MSC and activation were assessed weekly. Bayesian structural equation modeling revealed small effects of office-yoga (d = − 0.26) and walking (d = − 0.31) in reducing the prevalence of MSC as well as a program-based effectiveness of walking over office-yoga in reducing the intensity of MSC (d = 0.23). Concerning longitudinal trends, latent growth modeling revealed an intervention-based effectiveness for both office-yoga and walking on prevalence (doffice−yoga = − 0.32; dwalking = − 0.30) and intensity of MSC (doffice−yoga = − 0.42; dwalking = − 0.35). Neither intervention showed improvements in activation. We found no indication of adverse effects of either intervention. While both office-yoga and walking can effectively improve employees’ MSC, our findings suggest that walking is more effective in reducing intensity of MSC compared to office-yoga.