BACKGROUND: Circulating number of endothelial progenitor cells (EPCs) and EPC colony forming units (EPC-CFUs) have emerged as valuable predictors of cardiovascular health. Improvements in EPC number and EPC-CFUs have been associated with exercise and exercise training, although the mechanism for these improvements has not been identified. PURPOSE: This study examined the association of exercise-induced thrombin production with changes in circulating EPC number and EPC-CFUs as well as investigated the effects of exercise and thrombin supplementation on EPC differentiation and proliferation in vitro through gene expression analysis. METHODS: Subjects included healthy male Masters athletes (n = 12) and sedentary matched controls (n = 11) aged 55 - 80 years. Circulating EPC number, EPC-CFUs, plasma prothrombin fragment (F1+2) concentration and plasma thrombin-antithrombin III (TAT) concentration were measured at rest and after 30 minutes of vigorous treadmill exercise. Gene expression was assessed on resting EPC samples treated with 0U, 1U, 5U, or 10U of thrombin, as well as on EPC samples obtained after exercise. Gene expression analysis was performed for cell cycle genes cyclin A2, cyclin D1, and p27, and for cell surface markers VE-Cadherin and VEGFR2. To investigate the association of long-term exercise training, all outcomes were compared between Masters athletes and sedentary controls. RESULTS: Plasma concentrations of F1+2 and TAT increased significantly after exercise, however, EPC number and EPC-CFUs did not change. Changes in plasma F1+2 concentration with exercise correlated with changes in EPC number and EPC-CFUs in Masters athletes and with EPC number in control subjects Expression of the cell cycle genes cyclin A2 and cyclin D1 increased with thrombin treatment, while expression of the cell cycle inhibitor p27 decreased, peaking at 5U of thrombin. No change in VE-Cadherin or VEGFR2 expression was observed in control subjects, however, expression increased at 1U thrombin treatment in Masters athletes. Similarly, EPCs isolated after exercise demonstrated increased expression in cyclin A2 and cyclin D1, decreased in p27, and showed no change in VE-Cadherin or VEGFR2. CONCLUSION: Elevated thrombin production during vigorous exercise may play an important role in the regulation of EPCs, specifically cellular proliferation through changes in expression of cell cycle genes.