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To describe the associations among moderate-to-vigorous physical activity (MVPA), fitness, sedentary behavior (SB), and insulin sensitivity (IS).Data were drawn from the baseline assessment of the QUALITY cohort, which included 630 white youth (aged 8–10 years at recruitment), with at least one obese biological parent. IS was measured by two fasting indices (insulin, homeostasis model assessment of insulin resistance) and an oral glucose tolerance test (OGTT)–based index (Matsuda IS index [Matsuda-ISI]). Fitness was measured by VO2peak; percent fat mass (PFM) was measured by dual-energy X-ray absorptiometry; 7-day MVPA was measured with accelerometry. SB indicators included average hours daily of self-report screen time (SBst), and average minutes daily at <100 counts/min from accelerometry (SBacc). Multivariable linear regression models were adjusted for age, sex, season, and puberty.MVPA and SBacc were independently associated with IS, but this was no longer statistically significant after accounting for PFM. SBst was negatively associated with IS in girls only, even after controlling for physical activity (PA), fitness, and adiposity; for each additional hour of SBst daily, IS decreased by 4.6–5.6% across all IS indices. Fitness was positively associated with IS (measured by Matsuda-ISI) after accounting for PA, SB, and PFM; for every 1 unit increase in VO2peak, Matsuda-ISI increased by approximately 1.0% (P < 0.05).In children with an obese parent, PA and SBacc are associated with IS, but this association is mediated by adiposity. SBst is negatively associated with IS in girls, beyond its known impact on adiposity. Finally, fitness is independently associated with better IS measured by OGTT.