The increased erythrocyte aggregation observed in diabetes mellitus is mainly due to changes in the balance between aggregating factors and anti-aggregating ones, like albumin. Since chronic hyperglycaemia results in protein glycation, we examined the effect of in vitro glycation of albumin on its anti-aggregating role with blood from 29 Type 1 diabetic patients and 29 healthy controls. After the addition of glycated and unglycated albumin, samples had a glycation level of 24% for healthy controls and 28% for diabetic patients. Erythrocyte aggregation was determined by the analysis of the light backscattered by a blood suspension. Erythrocytes from healthy controls suspended in glycated albumin had significantly higher rates of rouleaux formation (p < 0.01) than in unglycated albumin and increased cohesion of rouleaux (p < 0.05). The erythrocyte aggregation in diabetic patients underwent similar changes (p < 0.01). The time-resolved fluorescence of the single tryptophan residue was monitored to describe the changes in the conformational equilibrium of albumin. The lifetime data showed that the increases in the two lifetime components and in the relative proportion of the major lifetime are in agreement with a conformational change in albumin after glycation. Thus, the changes in albumin conformation could be responsible for the smaller hypoaggregating effect of glycated albumin.