The PFA-100 device is increasingly used for assessing platelet function. Its use to monitor anti-platelet therapy, like acetylsalicylic acid (ASA), has been described. In most studies single PFA-100 measurements were used. In this study, we evaluate the influence of ASA on duplicate measurements using collagen/epinephrine cartridges. Twelve healthy volunteers received a single dose of 160 mg ASA and 12 other healthy volunteers received 30 mg ASA during 10 days followed by 80 mg ASA during 10 days. PFA-100 measurements were performed in duplicate 1 and 24 h after the final intake of medication. The mean coefficient of variation of duplicate measurements before medication was 8.4% and at least two times higher after the intake of a single dose of 160 mg ASA or 30 mg ASA during 10 days. Per individual, huge differences between duplicate measurements were observed after ASA ingestion. Differences were less pronounced after ingestion of 80 mg ASA during 10 days, because six of 12 volunteers had a maximum PFA-100 value>300 s in both measurements. As a consequence, one should be cautious to use the PFA-100 to monitor ASA therapy in individual patients.