Background: In a controlled, randomized, prospective, open, comparative study we evaluated the platelet function of 112 platelet concentrates (PC) prepared by apheresis during 5-day storage. Material and Methods: In one group, 56 PC were prepared by blood cell separator CS-3000 Plus (Baxter GmbH) and the collecting chamber PLT 30™ with the Omnix™ system; in the second group, 56 PC were prepared by blood cell separator AS-104 (Fresenius AG). In order to assess the platelet function of PC, the following parameters have been investigated at the day of donation and after 48-hour and 120-hour storage: platelet reactivity according to the Grotemeyer method, index of platelet aggregation induced by ADP and collagen, respectively, and platelet count. Results: Due to an elevated number of hyperaggregable platelets in PC separated with the CS-3000 Plus, platelet reactivity of PC in case of separation with the CS-3000 Plus was higher (1.46 ± 0.9) compared to that of PC separated with the AS-104 (1.20 ± 0.40; p = 0.064). The collagen-induced platelet aggregation after 48-hour storage was significantly higher in PC obtained by the AS-104 separator compared to that of the CS-3000 Plus device (1.54 ± 0.36 versus 1.45 ± 0.36; p < 0.018). With an increase of 63.7%, the level of ADP-induced aggregation was higher in PC collected with the AS-104 than that found in PC obtained by the CS-3000 Plus (34.3%; p = 0.018). With both methods, ADP-induced aggregation only occurred on the day of donation. In PC prepared by AS-104, the reduction of platelets during 5-day storage was 133 × 103/μl (11%) compared with 294 × 103/μl (21%) in PC generated by the CS-3000 Plus. The volume of PC showed also differences between the two methods. Conclusion: This investigation demonstrates the necessity to define quality characteristics for PC regarding platelet function in order to optimize the separation process.