Activity-based working (ABW) provides office workers with a variety of indoor workspaces purposively designed to accommodate different tasks. Despite an increased use of ABW, studies focusing on its impact and the resultant office design are yet to follow suit. This paper addresses this knowledge gap by providing empirical evidence from studies conducted before and after relocation from contemporary open-plan offices to ABW spaces. Results from post-occupancy evaluation (POE) surveys (n = 896 responses), spot measurements of indoor environmental quality (IEQ) and step-count monitoring (one case study; n = 20 participants) before and after relocation are reported. A total of 10 workspaces participated (six combi and four ABW) in this study. Design features were documented and analyzed. While there were limited differences in the measured IEQ conditions between office layouts investigated here, ABW workspaces yielded significantly higher satisfaction results on key IEQ dimensions, perceived productivity and health. Office layout was also found as a significant (or nearly significant) predictor of occupants’ lightly active and sedentary time but did not affect occupants’ daily step counts and distance they travelled. These results highlight the significance and impact of office layout and human-centred approach to design on occupants’ satisfaction, perceived productivity and incidental physical activity opportunities.