BACKGROUND: Stored red blood cells (RBCs) accumulate biochemical and biophysical changes. Maximum storage duration is based on acceptable in vitro characteristics and 24-hour survival, but not RBC function. Relatively little is known about the impact of RBC storage duration on oxygenation and the microcirculation. STUDY DESIGN AND METHODS: Eight healthy subjects donated a double RBC apheresis, which were prestorage leukoreduced and processed in AS-3. Subjects were transfused 1 unit of RBCs at 7 and 42 days after blood collection. Measurements of percentage of tissue oxygenation in the thenar eminence muscle (StO2) and brain (SctO2) were recorded with Food and Drug Administration-cleared noninvasive devices. Sublingual microvascular blood flow (microcirculatory flow index [MFI]) was quantified before and after RBC transfusion using a video microscope. Raw electronic data for all measurements were analyzed by a blinded observer at a core laboratory. RESULTS: The only pre- versus posttransfusion change observed in measurements of SctO2, StO2, or MFI was a very small increase in SctO2, from 70.4 to 71.8 (means, p=0.032) at 7 days. There was no significant difference in the amount of pre-post change at 7 days versus 42 days for any of the measures. CONCLUSION: Transfusion of 1 unit of 42-day-stored RBCs to healthy subjects has no overt detrimental effect on tissue oxygenation or the microcirculation assessed by clinically available monitors.