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This cross-sectional study examined the mutual independent associations of sedentary behavior, lower intensity physical activity (LPA) and higher intensity physical activity (HPA) (an approximation of moderate to vigorous physical activity with cardiorespiratory fitness (CRF).Two thousand twenty-four participants were included from The Maastricht Study (mean ± SD age, 59.7 ± 8.1 yr; 49.6% men). With the activPAL3 activity monitor, we assessed sedentary time (ST), sedentary pattern variables (number of sedentary breaks, average sedentary bout duration, and number of prolonged sedentary bouts [≥30 min]), LPA, and HPA. CRF was calculated as maximum power output per kilogram body mass (Wmax·kg−1) estimated from a submaximal cycle ergometer test. Linear regression analyses and isotemporal substitution analyses were used to examine associations of ST, sedentary pattern variables, and HPA with CRF. Analyses were stratified by sex.One hour of ST per day was associated with a lower Wmax·kg−1: Bmen = −0.03 (95% confidence interval [CI], −0.05 to −0.01) and Bwomen = −0.02 (95% CI, −0.04 to 0.00), independent of HPA. No statistically significant associations between sedentary patterns variables and CRF were observed. LPA was associated with a higher Wmax·kg−1: Bmen = 0.12 (95% CI, 0.07–0.17) and Bwomen = 0.12 (95% CI, 0.07–0.18). HPA was associated with a higher Wmax·kg−1: Bmen = 0.48 (95% CI, 0.38–0.58) and Bwomen = 0.27 (95% CI, 0.18–0.36). Replacing ST with LPA (Bmen, 0.08; 95% CI, 0.03–0.14; Bwomen, 0.10; 95% CI, 0.05–0.16) or with HPA (Bmen, 0.49; 95% CI, 0.39–0.59; Bwomen = 0.28; 95% CI, 0.19–0.36), but not with standing was associated with higher CRF.Modest associations between sedentary behavior and CRF were observed. Replacing ST with LPA was associated with higher CRF, which could be of particular importance for individuals who cannot engage in HPA. Nonetheless, replacing ST with HPA was associated with greatest estimated change in CRF.