Objective To examine the association between sedentary behavior and cardiometabolic risk, while taking into account cardiorespiratory fitness (fitness) and physical activity. Participants and Methods We examined the association of sedentary behavior, physical activity, and fitness (exposure variables) to cardiometabolic biomarkers and metabolic syndrome (outcome measures) among a historic cohort (January 2, 1981, through October 16, 2012) of men. First, we estimated the association (cross-sectionally and longitudinally) of sedentary behavior along with physical activity and fitness to lipids and lipoproteins, glucose, blood pressure, and markers of adiposity, including body mass index, waist circumference, and body fat percentage. We then prospectively examined the effects of baseline sedentary time on the incidence of metabolic syndrome, while adjusting for physical activity, fitness, and other covariates in multivariate models. Results Multivariate analysis of baseline data revealed that in comparison with the reference group (≤9 h/wk of sedentary time), more sedentary behavior was significantly associated with a higher triglyceride level, a higher triglycerides-high-density lipoprotein cholesterol ratio, and a higher body mass index, waist circumference, and body fat percentage (P<.05 for trend), after adjusting for physical activity and covariates. When adjusting for fitness and covariates, prolonged sedentary time was only associated with a higher triglyceride-high-density lipoprotein cholesterol ratio (P=.02 for trend). Sedentary time was not associated with the incidence of metabolic syndrome in multivariate models. Longitudinal analyses revealed that a 1-metabolic equivalent increase in fitness was significantly (P<.05) associated with almost all biomarkers when adjusting for sedentary behavior, with little moderation observed. Conclusion The association between prolonged sedentary time and cardiometabolic biomarkers is markedly less pronounced when taking fitness into account. Further exploration of the effects of sedentary behavior on cardiometabolic risk is warranted in cohorts with available fitness data. Furthermore, our findings underscore the need to encourage achieving higher fitness levels through meeting physical activity guidelines to decrease disease risk factors. © 2014 Mayo Foundation for Medical Education and Research.