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Abstract Backgrounds The purpose of this study was to examine, in a sample of Japanese older adults, the associations of objectively-assessed sedentary behavior (SB) and physical activity (PA) with performance-based physical function. The isotemporal substitution (IS) approach was used to model simultaneously the effects of the specific activity being performed and the activity being displaced, in an equal time-exchange manner. Methods Among 287 older adults (65–84 years), we used accelerometers to identify the daily average time spent on SB (≤1.5 METs); light-intensity PA (LIPA) (>1.5 to <3.0 METs); and moderate- to vigorous-intensity PA (MVPA) (≥3.0 METs). Physical function was assessed using five performance-based measures: hand grip strength, usual and maximum gait speeds, timed up and go, and one-legged stance with eyes open. We employed three linear regression models – a single-activity model, a partition model, and an IS model – to assess the associations of SB, LIPA, and MVPA with each of the five measures of physical function. Results There were significant positive associations in the single-activity and partition models between MVPA and the measures of physical function (with the exception of hand grip strength). The IS models found that replacing SB or LIPA with MVPA was significantly and favorably associated with physical function measures. Conclusions These findings indicate that replacing small amounts of SB and LIPA with MVPA (such as 10 min) may contribute to improvements in older adults’ physical function.