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Background: Previous studies on physical activity (PA) and pelvic organ prolapse (POP) were largely limited to self-reported PA in athletes, soldiers, and women in postpartum. We aimed to investigate the association of accelerometer-measured PA and sedentary behavior with the risk of POP in middle-aged and elderly women. Methods: In this prospective cohort derived from the UK Biobank, the intensity and duration of PA and sedentary behavior were measured with wrist-worn accelerometers over a 7-day period in 2013–2015 for 47,674 participants (aged 42.8–77.9 years) without pre-existing POP. Participants were followed up until the end of 2022, during which incident POP was ascertained mainly by the electronic health records. Multivariable-adjusted Cox proportional hazards models and restricted cubic splines were used to assess the associations of interest. Isotemporal substitution models were applied to test the effects of substituting a type of activity with equivalent duration of others. Results: During a median follow-up of 8.0 years, 779 cases of POP were recorded. The duration of light-intensity PA (LPA) was positively whereas sedentary time was negatively associated with the risk of POP. Every additional 1 h/day of LPA elevated the risk of POP by 18% (95% confidence interval [CI], 10%–26%). In contrast, the risk decreased by 5% (95% CI, 0–8%) per 1 h/day increment in sedentary behavior. No associations were found between moderate-intensity PA (MPA) or vigorous-intensity PA (VPA) and POP, except that women who had a history of hysterectomy were more likely to develop POP when performing more VPA (53% higher risk for every additional 15 min/day). Substituting 1 h/day of LPA with equivalent sedentary time was associated with a 18% (95% CI, 11%–24%) lower risk of POP. The risk can also be reduced by 17% (95% CI, 7%–25%) through substituting 30 min/day of LPA with MPA. Conclusions: More time spent in LPA or less sedentary time was linked to an elevated risk of POP in middle-aged and elderly women, while MPA or VPA was not. Substituting LPA with equivalent duration of sedentary behavior or MPA may lower the risk of POP. [ABSTRACT FROM AUTHOR]