OBJECTIVES: Despite successful antiretroviral (ARV) therapy, people living with HIV (PLWH) may show signs of premature/accentuated aging. We compared established biomarkers of aging in PLWH, appropriately-chosen HIV-negative individuals, and blood donors, and explored factors associated with biological age advancement. DESIGN: Cross-sectional analysis of 134 PLWH on suppressive ARV therapy, 79 lifestyle-comparable HIV-negative controls aged ≥45 years from the Co-morBidity in Relation to AIDS (COBRA) cohort, and 35 age-matched blood donors (BD). METHODS: Biological age was estimated using a validated algorithm based on ten biomarkers. Associations between 'age advancement' (biological minus chronological age) and HIV status/parameters, lifestyle, cytomegalovirus (CMV), hepatitis B (HBV) and hepatitis C virus (HCV) infections were investigated using linear regression. RESULTS: The average (95% CI) age advancement was greater in both HIV-positive [13.2 (11.6, 14.9) years] and HIV-negative [5.5 (3.8, 7.2) years] COBRA participants compared to BD [-7.0 (-4.1, -9.9) years, both p's < 0.001)], but also in HIV-positive compared to HIV-negative participants (p < 0.001). Chronic HBV, higher anti-CMV IgG titer and CD8 T-cell count were each associated with increased age advancement, independently of HIV-status/group. Among HIV-positive participants, age advancement was increased by 3.5 (0.1, 6.8) years among those with nadir CD4 < 200 cells/μL and by 0.1 (0.06, 0.2) years for each additional month of exposure to saquinavir. CONCLUSIONS: Both treated PLWH and lifestyle-comparable HIV-negative individuals show signs of age advancement compared to BD, to which persistent CMV, HBV co-infection and CD8 T-cell activation may have contributed. Age advancement remained greatest in PLWH and was related to prior immunodeficiency and cumulative saquinavir exposure