Exercise stimulates the release of hematopoietic and endothelial progenitor cells (EPC) from the bone marrow. However, no data are available concerning the time frame of EPC release during strenuous exercise. The aim of the present study was to investigate the time-dependent release of progenitor cells during strenuous exercise. Eighteen healthy young men cycled for 4 h continuously at 70% of their individual anaerobic threshold. Peripheral blood was drawn at 16 predefined time points during and after finishing cycling. A significant rise in heart rate and leukocytes was obvious, whereas lactate levels and hematocrit did not change. The amount of circulating progenitor cells, EPCs, mature endothelial cells (mECs), and microparticles, quantified by flow cytometry, showed a significant time-dependent increase at 210/240 min. In addition a very early rise in VEGF and later increase in IL-6, both measured by ELISA, were evident. All observed changes were normalized 24 h after finishing the test. In conclusion, strenuous activity in healthy individuals leads to a time-dependent increase in mECs, PCs, and EPCs that may be related to VEGF and IL-6.