As for other tests of hemostasis, the investigation of platelet function is highly vulnerable to a broad series of preanalytical variables, which span from patient preparation to the final analysis of the specimen and issuance of test results. In particular, there remains much controversy about the influence of manual or vacuum aspiration of blood into primary collection tubes on platelet function testing. Accordingly, we investigated this for the PFA-100. In 12 healthy volunteers, a sample labeled as ‘BD-V’ was drawn into a 2.7 ml BD Vacutainer tube, whereas two additional samples were collected from the opposite arm into 5.0 ml Sarstedt S-Monovette tubes by vacuum (SD-V) or manual aspiration (SD-A). All sample were tested on PFA-100 with collagen and ADP (CADP) or collagen and epinephrine (CEPI). The values of both CEPI and CADP obtained in SD-A samples were significantly lower than those obtained in SD-V and BD-V tubes, whereas those of the two evacuated tubes did not significantly differ. On average, CEPI values were prolonged by 11% in SD-V and 13% in BD-V, whereas those of CADP were prolonged by 14% in SD-V and 10% in BD-V, respectively. These findings suggests that the lower shear stress generated by the manual aspiration of blood into the primary collection tube would prevent spurious hyper-activation of platelets, thus, preserving the integrity of their function for subsequent testing on PFA-100. This study underscores the need to define or validate local reference ranges for the PFA-100 based on the collection tube used. Different reference ranges of both CEPI and CADP may also be advisable when venous blood samples are collected with manual aspiration or vacuum principle.