Abstract Background The duration of red blood cell (RBC) storage may have a negative impact on endothelial nitric oxide bioavailability. We tested the hypothesis that transfused fresh blood will have a more favourable effect on microvascular endothelial function as compared to older standard issue blood. Methods Participants requiring chronic RBC transfusions were enrolled in a crossover-design study to receive fresh (less than 7 days storage) or standard (up to 42 days storage) blood on two separate visits. Endothelial function was assessed by Reactive Hyperaemia Peripheral Arterial Tonometry (Rh-PAT) that was measured prior to and following transfusions. For each participant, the difference between endothelial function pre and post transfusion was assessed in relation to blood storage time. Results Twenty one patients (71 ± 16 years, 52% females) were enrolled. Mean age of fresh blood was 5.5 days ±1.0 and of standard blood was 24.5 days ±7.9 days. The pre-transfusion haemoglobin was 83.1 ± 2.5 g/L and post-transfusion 98.9 ± 2.6 g/L. An average of 2 units of packed Red Blood Cells ( pRBCs) were transfused. Microvascular endothelial function decreased more frequently after transfusion of standard blood compared to fresh blood. Standard issue blood transfusion was associated with decrease in Rh-PAT index (−0.25 ± 0.63) compared to fresh blood (+0.03 ± 0.49), P = .026. Conclusion Transfusions of standard issue blood is associated with less favourable effect on microvascular endothelial function as compared to fresh blood.