Background: International regulations for blood donation recommend a maximum donor age of 65 years. As the average population age is steadily rising in western societies, a considerable group of volunteers is lost to the donor base. Study Design and Methods: In a prospective study we investigated the effect of a 450-ml whole blood donation on the physical fitness and hemorheology of regular elderly allogeneic blood donors (n = 24, aged 63-69 years, mean = 65). Results were compared with a younger group of regular donors (n = 23, aged 55-62 years, mean = 58) and a group of elderly subjects (n = 7, aged 63-66 years, mean = 65), who did not donate blood for this study. Assessing the physical fitness, we determined the submaximal physical working capacity at a heart rate of 130 min-1 (PWC 130) and the maximal working capacity (MWC) by treadmill exercise testing the day before (day -1) and after donation (day +1). The impact of the blood loss on hemorheology was examined by analyzing the plasma viscosity before, during and after donation. Results: We found an increase of mean values of PWC 130 and MWC on day +1 in all study groups, but increases were only significant in the younger group (PWC 130 p = 0.03; MWC p = 0.04). Values did not differ significantly between the three groups. Plasma viscosity decreased significantly directly after donation in both groups of donors. Conclusion: A single blood donation did not alter the physical fitness of otherwise healthy elderly people. The older blood donors and the younger controls showed a similar compensation mechanism to blood loss. We found no general reason for disqualifying blood donors aged 65 years from donating.