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The detrimental effects of sedentary time on health may act by replacing time spent in physical activities. The aim of this study was to examine cross-sectional associations between objectively assessed sedentary and physical activity domains and cardiometabolic risk factors using a novel isotemporal substitution paradigm.Participants were 445 healthy men and women (mean age, 66 ± 6 yr), without history or objective signs of cardiovascular disease, drawn from the Whitehall II epidemiological cohort. Physical activity was objectively measured using accelerometers (ActiGraph GT3X) worn around the waist during waking hours for 4–7 consecutive days. We examined the effects of replacing sedentary time with light activity or moderate-to-vigorous physical activity (MVPA) on a range of risk factors (HDL cholesterol, triglycerides, HbA1c, and body mass index) using an isotemporal substitution paradigm.In partition models, where the time in each of the intensity categories was held constant, only MVPA remained associated with risk factors. In isotemporal substitution models that held total (wear) time constant, replacing 10-min sedentary time with an equal amount of MVPA was associated with favorable effects in all risk factors, including HbA1c (B = −0.023; 95% confidence interval (CI), −0.043 to −0.002), BMI (B = −0.39; 95% CI, −0.54 to −0.24), HDL cholesterol (B = 0.037; 95% CI, 0.021–0.054), and triglycerides (B = −0.035; 95% CI, −0.061 to −0.009).The associations between sedentary behavior and cardiometabolic risk may be dependent on the types of activities that are displaced by sedentary time.