The positive association between the total duration of physical activity and performances of physical function may vary at different times of the day as circadian rhythm regulates individuals in response to external stimulations. We aimed to examine the association of timing-specific and overall moderate-to-vigorous physical activity (MVPA) with performances of physical function in older adults. A cross-sectional analysis was conducted among 118 older adults (mean age = 70.0 ± 5.0 years). We assessed and identified timing-specific (morning: 06:01-12:00; afternoon: 12:01-18:00; evening: 18:01-24:00) and overall MVPA using a triaxial accelerometer. Different measures of physical function were evaluated including handgrip strength (by grip dynamometer), gait speed (5-m walk test), basic functional mobility (timed up and go test), and lower limb strength (five times sit-to-stand test). Multivariate linear regression models adjusting for covariates were used to investigate the associations. Participants spent 25.0 (± 26.2) minutes in MVPA per day on average, half the time spent during the morning (47.7%), followed by during the afternoon (29.9%) and evening (21.6%). The time spent on overall MVPA was generally associated with better physical function performances. There was statistical evidence for the percentages of MVPA engagement during the morning [B = 0.214, 95% confidence interval (CI) 0.001 to 0.428] and afternoon (B = - 0.273, 95% CI - 0.518 to - 0.027) associated with basic functional mobility but with contrary directions; the percentage of MVPA engagement during the evening was associated with less time spent in gait speed performance (B = - 0.237, 95% CI - 0.468 to - 0.006). Our findings inform implications that the overall MVPA engagement was more important than timing-specific MVPA to older adults' physical function performances. Strategies for accumulating time of MVPA is more practical and effective than encouraging to engage MVPA in specific timing for the enhancement of functional ability and therefore prevent disability among older adults.