Background: Ageing is associated with a decline in immune function termed immunosenescence. This process is characterized amongst others by less naive T-cells and more senescent phenotypes, which have been implicated in the pathogenesis of many age-related diseases. Thus far, reports regarding the long-term adaptation effects of exercise on T-cell phenotypes are scant and largely equivocal. These inconsistencies may be due to potential contributors to immunosenescence, particularly cytomegalovirus infection, which is considered a hallmark of T-cell senescence. Therefore, we sought to investigate the impact of cytomegalovirus serostatus on the distribution of peripheral T-cell subsets following long-term exercise in older women. Methods: One hundred women (aged 65 years and above) were randomized to 3 times/weekly training at either intensive strength training (3 × 10 repetitions at 80% of one-repetition maximum, n = 31), strength endurance training (2 × 30 repetitions at 40% of one-repetition maximum, n = 33), or control (passive stretching exercise, n = 36) for 6 weeks. All training sessions were supervised by trained instructors to minimize the risk of injury and to ensure that the participants adhered to the training protocol throughout the entire range of motion. The T-cell percentages and absolute blood counts were determined before and after 6 weeks (24 h-48 h after the last training session) using flow cytometry and a haematology analyser. Cytomegalovirus antibodies were measured in serum using Architect iSystem and cytomegalovirus serostatus was balanced in the three intervention groups. C-reactive protein was measured using immunonephelometry. Results: We report for the first time that 6 weeks of strength endurance training significantly decreased senescence-prone T-cells along with a small increase in the number of CD8- naive T-cells in blood. The absolute counts of senescent-like T-cells decreased by 44% (from 26.03 ± 35.27 to 14.66 ± 21.36 cells/μL, p < 0.01) and by 51% (from 6.55 ± 12.37 to 3.18 ± 6.83 cells/μL, p < 0.05) for the CD8+ and CD8- T-cell pools, respectively. Intriguingly, these changes were observed in cytomegalovirus seropositive, but not cytomegalovirus seronegative individuals. Conclusions: In conclusion, the present study shows that strength endurance training leads to a reduction in circulating senescence-prone T-cells in cytomegalovirus seropositive older women. It remains to be established if monitoring of peripheral senescence-prone T-cells may have utility as cellular biomarkers of immunosenescence.