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Data evaluating mortality benefit from replacing sedentary time with physical activity are sparse. We explored reallocating time spent in sedentary behavior to physical activity of different intensities in relation to mortality risk.Women and men age 50–85 yr from the National Health and Nutrition Examination Survey 2003–2004 and 2005–2006 cycles with follow-up through December 31, 2011, were included. Sedentary time and physical activity were assessed using an ActiGraph accelerometer. Isotemporal substitution models were used to estimate the effect of replacing one activity behavior with another activity behavior for the same amount of time while holding total accelerometer wear time constant.During a mean follow-up of 6.35 yr, 697 deaths from any cause occurred. Replacing 30 min of sedentary time with an equal amount of light activity was associated with 14% reduced risk of mortality (multivariable-adjusted hazard ratio (HR), 0.86; 95% confidence interval (CI), 0.83–0.90). Replacement of sedentary time with moderate to vigorous activity was related to 50% mortality risk reduction (HR, 0.50; 95% CI, 0.31–0.80). We also noted a 42% reduced risk of mortality when light physical activity was replaced by moderate to vigorous activity (HR, 0.58; 95% CI, 0.36–0.93).Replacing sedentary time with an equal amount of physical activity may protect against preterm mortality. Replacement of light physical activity with moderate to vigorous activity is also associated with protection from premature mortality.