Combination antiretroviral therapy (ART) has markedly improved survival in HIV-infected patients, but not without significant adverse effects including ART-associated dyslipdemia and insulin resistance, which may in part contribute to an increased risk of cardiovascular events. Other contributing factors to cardiovascular risk may include uncontrolled HIV replication, the effects of HIV and ART on vascular endothelium and inflammatory cytokines. Diastolic dysfunction may be an early sign of cardiovascular disease. Our objective was to determine the prevalence of diastolic dysfunction in HIV-infected patients without cardiovascular symptoms. We enrolled 91 subjects in a cross-sectional study of HIV-infected patients without cardiovascular symptoms between September 2004 and August 2005, to assess whether demographics, HIV-related factors, cardiac risk factors, and ART were associated with diastolic dysfunction. All subjects underwent two-dimensional transthoracic echocardiography with tissue Doppler imaging. Subjects were predominately male with a median age of 38 (interquartile range [IQR]: 33, 42) years and median ART duration 6.15 (IQR: 2.1, 8.4) years. Subjects had low Framingham risk scores. Diastolic dysfunction was observed in 34 patients (37%; 95% confidence interval [CI] 27.4, 48.1). Cardiac risk factors or poor prognostic indicators of AIDS progression were uncommon with no difference between subjects with or without diastolic dysfunction. A nonstatistically significant trend in increased rate of diastolic dysfunction was observed in patients receiving protease inhibitors 1 year or more, 44% versus 28%, respectively (univariate odds ratio 2.02, 95% CI 0.83 to 4.90). This was not observed with prolonged use of either non-nucleoside or nucleoside reverse transcriptase inhibitors. A high prevalence of diastolic dysfunction (37%) in a cohort of HIV-infected patients on ART at low risk for AIDS and cardiovascular disease was demonstrated.