This cross-sectional study is one of the first to examine and compare the independent associations of objectively measured sedentary time, moderate to vigorous physical activity (MVPA) and fitness with cardio-metabolic risk factors. We studied 543 men and women (aged 18-49 years) from the NHANES 2003-2004 survey. Sedentary time and MVPA were measured by accelerometry. Fitness was assessed with a submaximal treadmill test. Cardio-metabolic risk factors included: waist circumference (WC), BMI, blood pressure, fasting glucose, HDL- and non HDL cholesterol, triglycerides (TG), and C-reactive protein (CRP). Sedentary time, MVPA and fitness were used as predictors for the cardio-metabolic outcomes in a multiple regression analysis. Standardized regression coefficients were computed. Results show that sedentary time was associated with HDL-cholesterol (β=-0.080, p=0.05) and TG (β=0.080, p=0.03). These results became non-significant after adjustment for MVPA and fitness. MVPA was associated with WC (β=-0.226), BMI (β=-0.239), TG (β=-0.108) and HDL-cholesterol (β=0.144) (all p<0.05). These results remained significant after adjustment for sedentary time and fitness. Fitness was associated with WC (β=-0.287), BMI (β=-0.266), systolic blood pressure (β=-0.159), TG (β=-0.092), and CRP (β=-0.130) (all p<0.05). After adjustment for sedentary time and MVPA these results remained significant. These differences in relative importance of sedentary time, MVPA and fitness on cardio-metabolic-risk are important in the design of prevention programs. In this population, the strength of the associations between MVPA and fitness with cardio-metabolic markers appeared to be similar; both MVPA and fitness showed independent associations with cardio-metabolic risk factors. In contrast, sedentary time showed no independent associations with cardio-metabolic risk after correction for fitness and MVPA.