Objectives: Despite successful antiretroviral 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 antiretroviral therapy, 79 lifestyle-comparable HIV-negative controls aged 45 years or older from the Co-morBidity in Relation to AIDS (COBRA) cohort, and 35 age-matched blood donors. Methods: Biological age was estimated using a validated algorithm based on 10 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 with blood donors [-7.0 (-4.1 to -9.9) years, both P's< 0.001)], but also in HIV-positive compared with HIV-negative participants (P< 0.001). Chronic HBV, higher anti-CMV IgG titer and CD8 thorn 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 thorn T-cell count less than 200 cells/ ml and by 0.1 (0.06-0.2) years for each additional month of exposure to saquinavir. Conclusion: Both treated PLWH and lifestyle-comparable HIV-negative individuals show signs of age advancement compared with blood donors, 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.