Non-responsiveness to aspirin as detected by laboratory tests may identify patients at high risk for future vascular events. The aim of this prospective study was to evaluate whether non-responsiveness to aspirin is stable over time. Ninety-eight patients with stable peripheral arterial occlusive disease (PAOD) treated with 100 mg/d aspirin were followed over a median timeframe of 17 months. Platelet function tests were performed initially and at follow-up using arachidonic acid-induced light transmittance aggregometry (LTA) in native platelet-rich plasma with the Behring Coagulation Timer and by measuring the collagen-epinephrine closure time (CT) on a Platelet Function Analyzer (PFA-100). When determining platelet function using LTA, four patients (4.1%) had residual platelet function (i.e., MaxAggr > or =78%) despite aspirin treatment, whereas, according to the PFA-100 results, 12 patients (12.2%) were identified as non-responders (i.e., CT <192 s). Fifty-seven patients who were still under treatment with 100 mg/d aspirin at the time of follow-up provided a second blood sample. Further platelet function tests with the PFA-100 system identified a persistent non-responsiveness to aspirin over time in three patients (5.3%) whereas four (7.0%) and 15 (26.3%) patients had changes in response status when platelet function was assessed by LTA and on the PFA-100(R), respectively. We conclude that true non-responsiveness to aspirin is a rare phenomenon in stable PAOD patients. Furthermore, we conclude that in a number of patients, aspirin non-responsiveness is not stable over time.