Plasma-reduced platelet concentrates are commonly administered to prevent febrile transfusion reactions and to avoid fluid overload in neonates. Because little is known about the influence of centrifugation and resuspension on functional aspects of platelets, we examined the effects of plasma-reduction on platelet aggregation and platelet-dependent thrombin generation. Our results show that plasma reduction and resuspension of the platelet pellet in saline or plasma results in a significant reduction in platelet aggregation to a combination of the platelet agonists adenosine diphosphate and epinephrine (p < 0.001). In contrast, when a combination of the more potent agonists collagen and thrombin was used, platelet aggregation was maintained. Likewise, no decline was observed in platelet-dependent thrombin generation as measured by the functional prothrombinase assay or Annexin V binding. We conclude that centrifugation and resuspension of platelets to render the concentrate plasma-free, as a routine procedure in blood banking, variably affects in vitro platelet aggregability but does not significantly affect platelet-dependent thrombin generation.