During their 120-day life span, human red blood cells (RBC) undergo several physicochemical changes, including an increased tendency to aggregate in plasma or polymer solutions. This study was designed to examine potential associations between age-related differences in RBC mobility, aggregation, and membrane glycocalyx properties for cells suspended in buffer and in 3 g/dl solutions of 70.3 kDa dextran. A recent model for depletion-mediated RBC aggregation was employed to calculate the changes of glycocalyx properties that were consistent with experimental electrophoretic mobility (EPM) and aggregation data. Young and old cells were obtained by density separation, after which aggregation and EPM were determined versus ionic strength; old cells exhibited a two- to threefold greater aggregation in dextran. EPM of old cells was identical to young cells in polymer-free media yet was 4% greater in dextran. The greater EPM for old RBC indicates a larger polymer depletion layer, which could be explained either by a 10-15% decrease of their glycocalyx thickness or a similar percentage decrease of polymer penetration into their glycocalyx. The larger depletion layer leads to markedly elevated cell-cell affinities for old cells, with the computed affinity increases consistent with enhanced old RBC aggregation. These results provide a rational explanation for the aggregation and EPM behavior of old RBC, and raise the possibility of depletion-mediated interactions contributing to senescent cell removal from the circulation.