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This study aimed to examine the associations between objectively measured physical activity (PA), sedentary time, and health-related fitness and to investigate the combined association of PA and sedentary time on health-related fitness in youths.PA and sedentary time were assessed with accelerometers in 2506 youths age 10–18 yr (Mage = 13.2 ± 2.3). Participants were classified as active (≥60 min·d−1 of moderate-to-vigorous physical activity (MVPA)) versus inactive (<60 min·d−1 of MVPA) and as “low sedentary” versus “high sedentary” (according to the median value of sedentary time per day) and thereafter grouped as active/low sedentary, active/high sedentary, inactive/low sedentary, and inactive/high sedentary. Five physical fitness tests (body mass index, push-ups, curl-ups, sit and reach, and the Progressive Aerobic Cardiovascular Endurance Run test) were assessed with FITNESSGRAM, and participants were categorized as being in the healthy fitness zone (HFZ) versus the unhealthy fitness zone. A fitness composite score was calculated using the individual fitness test z-score. Regression models were used to examine the relation between PA, sedentary time, and physical fitness.Time spent in MVPA (min·d−1) (β = 0.002, P < 0.001) was significantly associated with fitness score independent of sedentary time. Sedentary time was not associated with physical fitness independent of MVPA. Compared with the inactive/high sedentary group (referent), being categorized as active/low sedentary was associated with increased likelihood of being in the HFZ for sit and reach (odds ratio, 2.55; 95% confidence interval, 1.96–3.32) and having a higher fitness composite score (odds ratio, 1.38; 95% confidence interval, 1.13–1.69).Time in MVPA was associated with better physical fitness independent of sedentary time. Participants classified as active/low sedentary had higher odds of being in the HFZ, for flexibility, and to have a better fitness composite score. These findings suggest that time in MVPA contributes to better physical fitness in youths.