Background: Low physical activity is widespread and poses a serious public health challenge both globally and in the UK. The need to increase population levels of physical activity is recognised in current health policy recommendations. There is considerable interest in whether or not the built environment influences health behaviours, particularly physical activity levels, but longitudinal evidence is limited. Objectives: The effect of moving into East Village (the former London 2012 Olympic and Paralympic Games Athletes' Village, repurposed on active design principles) on the levels of physical activity and adiposity, as well as other health-related and well-being outcomes among adults, was examined. Design: The Examining Neighbourhood Activities in Built Environments in London (ENABLE London) study was a longitudinal cohort study based on a natural experiment. Setting: East Village, London, UK. Participants: A cohort of 1278 adults (aged ≥ 16 years) and 219 children seeking to move into social, intermediate and market-rent East Village accommodation were recruited in 2013-15 and followed up after 2 years. Intervention: The East Village neighbourhood, the former London 2012 Olympic and Paralympic Games Athletes' Village, is a purpose-built, mixed-use residential development specifically designed to encourage healthy active living by improving walkability and access to public transport. Main outcome measure: Change in objectively measured daily steps from baseline to follow-up. Methods: Change in environmental exposures associated with physical activity was assessed using Geographic Information System-derived measures. Individual objective measures of physical activity using accelerometry, body mass index and bioelectrical impedance (per cent of fat mass) were obtained, as were perceptions of change in crime and quality of the built environment. We examined changes in levels of physical activity and adiposity using multilevel models adjusting for sex, age group, ethnic group, housing sector (fixed effects) and baseline household (random effect), comparing the change in those who moved to East Village (intervention group) with the change in those who did not move to East Village (control group). Effects of housing sector (i.e. social, intermediate/affordable, market-rent) as an effect modifier were also examined. Qualitative work was carried out to provide contextual information about the perceived effects of moving to East Village. Results: A total of 877 adults (69%) were followed up after 2 years (mean 24 months, range 19-34 months, postponed from 1 year owing to the delayed opening of East Village), of whom 50% had moved to East Village; insufficient numbers of children moved to East Village to be considered further. In adults, moving to East Village was associated with only a small, non-significant, increase in mean daily steps (154 steps, 95% confidence interval -231 to 539 steps), more so in the intermediate sector (433 steps, 95% confidence interval -175 to 1042 steps) than in the social and market-rent sectors (although differences between housing sectors were not statistically significant), despite sizeable improvements in walkability, access to public transport and neighbourhood perceptions of crime and quality of the built environment. There were no appreciable effects on time spent in moderate to vigorous physical activity or sedentary time, body mass index or percentage fat mass, either overall or by housing sector. Qualitative findings indicated that, although participants enjoyed their new homes, certain design features might actually serve to reduce levels of activity. Conclusions: Despite strong evidence of large positive changes in neighbourhood perceptions and walkability, there was only weak evidence that moving to East Village was associated with increased physical activity. There was no evidence of an effect on markers of adiposity. Hence, improving the physical activity environment on its own may not be sufficient to increase population physical activity or other health behaviours. Funding: This project was funded by the National Institute for Health Research (NIHR) Public Health Research programme and will be published in full in Public Health Research; Vol. 8, No. 12. See the NIHR Journals Library website for further project information. This research was also supported by project grants from the Medical Research Council National Prevention Research Initiative (MR/J000345/1).