Background Transfusion of storage-aged (aged) packed red blood cells (pRBCs) is associated with worse outcomes, but the underlying mechanisms remain unknown. We measured plasma nitrite (NO2) levels and tested brachial vascular reactivity during transfusion of aged and fresh pRBCs with the hypothesis that aged pRBCs will impair NO2 and endothelial function. Methods Forty-four hospital inpatients (aged 59 ± 14 years, 46% female) receiving cross-matched pRBC transfusions for clinical indications were randomized to receive either fresh (aged 21 days, n = 24). NO2 was measured via high-performance liquid chromatography and vascular endothelial function was assessed using flow-mediated dilation (FMD) of the brachial artery before, during, and following (1 and 24 h) transfusion. Data were analyzed using a linear mixed effects model. Results There were no differences in demographics, CVD risk factors, known CVD, amount of pRBC transfused, or baseline NO2 or FMD between groups. The mean (±SD) age of pRBC units was 9.1 ± 3.1 days and 29.8 ± 5.7 days in the fresh and aged groups, respectively. Patients receiving fresh blood had a 0.191 ± 0.438 and 0.069 ± 0.242 μM increase in NO2 at 1 and 24 h after transfusion, respectively, whereas, patients receiving aged blood experienced a 0.055 ± 0.227 and 0.079 ± 0.226 μM decrease in NO2 at 1 and 24 h representing a difference in NO2 response between fresh and aged blood, p = 0.027 and p = 0.052 at 1 and 24 h, respectively (Panel A). Patients receiving fresh pRBCs had no significant change in FMD. However, FMD significantly decreased by 2.4% (p = 0.037) 24 h after transfusion of aged pRBCs (Panel B). Summary and conclusions Transfusion of aged, but not fresh blood is associated with decreased plasma NO2 and FMD response within 24 h of transfusion. Aged transfusion-induced endothelial dysfunction may contribute to poor outcomes associated with blood transfusion. Strategies to improve NO bioactivity in transfusion need to be further investigated. Disclosure Nothing to disclose.