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To assess whether mitochondrial dysfunction in skeletal muscle characterizes antiretroviral therapy (ART)-associated lipoatrophy (LA).A cross-sectional study comparing HIV-infected, antiretroviral-treated patients with LA (n = 5; LA+) and without LA (n = 5; non-LA) was conducted. Positron emission tomography was used to measure blood flow, oxygen extraction and oxygen consumption in quadriceps femoris muscle during rest and aerobic exercise. Mitochondrial DNA (mtDNA) was quantified by PCR. Body composition was measured by dual-energy X-ray absorptiometry and magnetic resonance imaging. All data are given as means ± SEM.Compared with the non-LA group, the LA+ group had significantly less limb fat and more intra-abdominal fat, but similar leg muscle mass. The LA+ group versus the non-LA group had reduced mtDNA content per nucleus in adipose tissue (173 ± 38 versus 328 ± 62; P = 0.067), but not in skeletal muscle (2606 ± 375 versus 2842 ± 309; P = 0.64). Perfusion in resting muscle (34 ± 7 versus 28 ± 6 mL/kg/min in the LA+ group versus the non-LA group; P = 0.5), and the mean absolute (277 ± 30 versus 274 ± 43 mL/kg/min, respectively; P = 0.95) and relative (10.6 ± 2.5- versus 11.9 ± 1.5-fold change, respectively; P = 0.67) increases in perfusion during exercise were similar between the groups. Oxygen consumption at rest (2.2 ± 0.7 versus 2.1 ± 0.3 mL/kg/min in the LA+ group versus the non-LA group; P = 0.9), and the mean absolute (14.6 ± 1.7 versus 24.3 ± 8.8 mL/kg/min, respectively; P = 0.3) and relative (10.3 ± 2.8- versus 11.7 ± 2.4-fold change, respectively; P = 0.73) exercise-induced increases in oxygen consumption were similar between the groups. The oxygen extraction fraction was comparable between the groups, both at rest and during exercise. Plasma lactate concentrations remained unchanged in both groups during exercise.HIV-infected patients with ART-associated LA have similar mtDNA content in skeletal muscle and comparable skeletal muscle aerobic exercise metabolism to antiretroviral-treated non-lipoatrophic patients.