Objective: To evaluate the long-term effects of anti-retroviral treatment (ART) interruptions on metabolic, immunological, virological and clinical outcomes in chronically HIV-1 infected patients. Methods: Multi-centric, prospective, controlled 24-month cohort study in HIV-1 infected patients interrupting ART once or several times and for at least two weeks. Patients were compared to a frequency-matched control group continuing on ART. Results: A total of 399 HIV-1 infected patients were included, among them 133 patients with treatment interruption (TI) and 266 control patients. Baseline characteristics were well matched. Median baseline CD4 cell count was 379/mu l in TI-patients and 410/mu l in control patients (p = ns). Median duration of the first TI was 1.1 months, and 37 % of patients had two or further TI's. Whereas CD4 cell count in control patients had increased significantly by a median of 67/mu l at month 24 (p < 0.0001), median CD4 cell count at month 24 in the TI-patients did not differ significantly from base-line. However, two-year AIDS-free survival was not significantly different between TI- and control patients. Liver enzymes and blood lipids improved significantly during TI. Conclusion: TI was associated with a significant immunological disadvantage at 24-month follow-up compared to continued ART. In this relatively immunocompetent cohort, however, TI's did not lead to an increased risk of disease progression within two years of follow-up.