Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily (od), low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase (COX)-1 inhibition. We performed a multicenter, double-blind trial to investigate the efficacy of three aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Two-hundred-forty-five patients on chronic od low-dose aspirin were randomized (1:1:1) to receive 100 mg aspirin od, twice-daily, bid), or three-times daily (tid) for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate endpoints of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the bid and tid regimens showed substantially reduced inter-individual variability and lower median values of sTXB2: 19.3[9.7-40], 4 [2.1-6.7], and 2.5[1.4-5.65] ng/ml in the od (n=85), bid (n=79) and tid (n=79) arms, respectively. Urinary PGIM was comparable in the three arms. Urinary TXM was significantly reduced by 35% in both experimental arms. Patients in the tid arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75-100 od for cardiovascular prophylaxis appears largely inadequate in reducing platelet activation in the vast majority of ET patients. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement by further reducing it. (EudraCT 2016-002885-30).