Red blood cells (RBCs) capability to deliver oxygen (O2) has been routinely measured by P50. Although this defines the ability of RBCs to carry O2 under equilibrium states, it cannot determine the efficacy of O2 delivery in dynamic blood flow. Here, we developed a microfluidic analytical platform (MAP) that isolates single RBCs for assessing transient changes in their O2 release rate. We found that in vivo (biological) and in vitro (blood storage) aging of RBC could lead to an increase in the O2 release rate, despite a decrease in P50. Rejuvenation of stored RBCs (Day 42), though increased the P50, failed to restore the O2 release rate to basal level (Day 0). The temporal dimension provided at the single-cell level by MAP could shed new insights into the dynamics of O2 delivery in both physiological and pathological conditions.