Aim. There is evidence for the existence of a functional endothelial nitric oxide synthase (eNOS) isoform in human red blood cells (RBCs), which supports the hypothesis that RBCs, beside endothelial cells (ECs), display an important source of NO in the microvascular environment as eNOS was originally attributed to ECs. This enzyme is critically involved in the generation of nitric oxide (NO). NO plays a crucial role in the mechanisms inducing RBC deformability and subsequently contribute to the microvascular rheology. Until today it remains to be elucidated whether there is a direct link between RBCs' deformability capacities, eNOS activation and NO release in RBCs induced by physical exercise. Methods. Six male, healthy, recreationally active subjects (age [yrs]: 29 ± 2.7) participated in this study. The subjects performed two different running test: an incremental running test (IRT) (start: 2.0 m*sec–1, increase 0.5 m*sec–1 every 5 min) and a 60 min continuous running test (CRT) (70% VO2max). Each of the following parameters was measured before and after exercise: elongation index (EI) of RBCs (by diffraction ectacytometry); eNOS, peNOSSer1177 (by immunohistochemistry); NO formation in RBCs (by DAF-2DA and laser scan microscopy). Results. EI, eNOS, peNOSSer1177, and NO were upregulated after IRT compared to basal levels (p<.05). Regarding CRT only the data for EI and NO are available until today. In accordance to the data from IRT, EI and NO were increased after CRT compared to basal level (p<.05). Conclusion. The presented data show that physical exercise has a beneficial effect on EI of RBCs. It is demonstrated that eNOS is activated and can be involved in this process by inducing elevated amounts of NO in RBCs. In addition, it is shown that exercise enhances NO formation in RBCs. Taken together the results provide important insight into the mechanisms of RBCs deformability and highlight positive effects of exercise.