For investigations of platelet function it is recommended that venipuncture should be performed using ordinary needle systems instead of butterfly cannulae systems. Platelets might be activated in the long plastic tubes of butterfly systems. The aim of this study was to investigate the dependency of platelet function results on blood sampling using different collection systems. Therefore, blood of 25 healthy volunteers was collected from both arms using at the same time on one side a 21-gauge needle and on the other side a 21-gauge butterfly cannula system. Both samples of each volunteer were analyzed on the PFA-100. Platelet aggregation was performed on the Behring Coagulation Timer (BCT) and the optical aggregometer PAP-4 using ADP, collagen and arachidonic acid to induce platelet aggregation in platelet-rich plasma. No significant prolongation of the closure times on the PFA-100 with the COL/EPI cartridge and the COL/ADP cartridge was observed when using butterfly cannulae. The results of optical aggregometry were not significantly different. The maximum aggregation response did not differ significantly for both collection systems. Aggregometry and the PFA-100 system are not affected by different blood collection systems. Therefore butterfly cannulae can be used for sample collection to investigate platelet function.