OBJECTIVES: Assess differences in movement behaviours within the 24-hour cycle, including light intensity physical activity (LPA), moderate-to-vigorous physical activity (MVPA), sedentary time and sleep, before and during the COVID-19 pandemic and assess these differences stratified by several relevant factors in a subcohort of the Cancer Prevention Study-3. DESIGN AND SETTING: US-based longitudinal cohort study (2018-August 2020). PARTICIPANTS: N=1992 participants, of which 1304 (65.5%) are women, and 1512 (75.9%) are non-Latino white, with a mean age 57.0 (9.8) years. MEASURES: Age, sex, race/ethnicity, education; self-reported LPA, MVPA, sedentary time and sleep duration collected before and during the pandemic; pandemic-related changes in work, childcare and living arrangement; COVID-19 health history. RESULTS: Compared to 2018, participants spent an additional 104 min/day sedentary, 61 fewer min/day in LPA and 43 fewer min/day in MVPA during the pandemic. Time spent sleeping was similar at the two time points. Differences in movement behaviours were more pronounced among men, those with a higher level of education, and those who were more active before the pandemic. CONCLUSIONS: From 2018 to Summer 2020, during the COVID-19 pandemic, US adults have made significant shifts in daily time spent in LPA, MVPA and sedentary. There is an urgent need to promote more physical activity and less sedentary time during this public health crisis to avoid sustaining these patterns long-term.