OBJECTIVES: To investigate the short- and long-term dynamics of intact and defective proviral HIV DNA during ART. DESIGN: We evaluated viral reservoir dynamics in a cohort of nine individuals with chronic HIV-1 subtype B infection who initiated first-line ART and were followed for 20 years while continuing ART. METHODS: PBMCs were obtained before ART (n = 5), during the first year, and after 8.5 and 20 years of treatment. T cell subsets (naive, central-memory, transitional-memory and effector-memory) were sorted at 8.5 and 20 years. DNA was isolated and analyzed using the intact proviral DNA assay (IPDA). Deep-sequencing of the viral env region enabled analysis of viral evolution and cellular mechanisms underlying HIV persistence. RESULTS: Initially, defective and intact proviral DNA in PBMCs declined with half-lives of 3.6 and 5.4 weeks, respectively. Over the following 8.5 years, the intact reservoir continued to decrease, with a half-life of 18.8 months in PBMCs, while defective proviral DNA levels stabilized. After 8.5 and 20 years of ART, the intact reservoir showed no further decline, with most intact proviral DNA residing in memory T cell subsets. Phylogenetic analysis revealed no signs of viral evolution over time, both within and between T cell subsets. CONCLUSIONS: PBMCs containing intact proviral DNA are selectively lost during the first decade of suppressive ART, followed by a decade of stabilization of this reservoir in the memory T cell subsets. In the absence of clear signs of viral evolution and massive clonal expansion, homeostatic proliferation might be an important driver of HIV persistence during long-term ART.