Obesity, sedentary lifestyles, and antiretroviral therapies may predispose HIV-infected children to poor physical fitness. Estimated peak oxygen consumption (VO(2) peak), maximal strength and endurance, and flexibility were measured in HIV-infected and uninfected children. Among HIV-infected children, anthropometric and HIV disease-specific factors were evaluated to determine their association with VO(2) peak. Forty-five HIV-infected children (mean age 16.1 years) and 36 uninfected children (mean age 13.5 years) participated in the study. In HIV-infected subjects, median viral load was 980 copies/ml (IQR 200-11,000 copies/ml), CD4% was 28% (IQR 15-35%), and 82% were on highly active antiretroviral therapy (HAART). Compared to uninfected children, after adjusting for age, sex, race, body fat, and siblingship, HIV-infected children had lower VO(2) peak (25.92 vs. 30.90 ml/kg/min, p<0.0001), flexibility (23.71% vs. 46.09%, p=0.0003), and lower-extremity strength-to-weight ratio (0.79 vs. 1.10 kg lifted/kg of body weight, p=0.002). Among the HIV-infected children, a multivariable analysis adjusting for age, sex, race, percent body fat, and viral load showed VO(2) peak was 0.30 ml/kg/min lower per unit increase in percent body fat (p<0.0001) and VO(2) peak (SE) decreased 29.45 (± 1 .62), 28.70 (± 1.87), and 24.09 (± 0.75) ml/kg/min across HAART exposure categories of no exposure, <60, and ≥ 60 months, respectively (p<0.0001). HIV-infected children had, in general, lower measures of fitness compared to uninfected children. Factors negatively associated with VO(2) peak in HIV-infected children include higher body fat and duration of HAART ≥ 60 months. Future studies that elucidate the understanding of these differences and mechanisms of decreased physical fitness should be pursued.