Objectives: Rotating shift work has been reported to increase the risk of cardiovascular diseases. Vascular endothelial dysfunction and platelet activation are among the leading causes of thrombus formation in patients with myocardial infarction or stroke. Endothelial function has been shown to be impaired immediately after night-shift work; however, it is not known whether platelets are also activated. The aim of this study was to investigate the acute impact of night-shift work on platelet function. Methods: This observational study included 11 healthy medical staff members (seven women, median age 32 years). We examined each subject's platelet aggregation rates and the serum concentrations of eicosanoid mediators after night-shift work and on day-shift work without preceding night-shift work (baseline). Results: Platelet aggregation did not differ from baseline levels after night-shift work. However, serum cyclooxygenase (COX)-metabolized eicosanoid mediators, particularly thromboxane (Tx) B₂ (a stable metabolite of TxA₂ and the most important marker of platelet activation), were significantly higher after the night-shift than at baseline (median 65.3 vs 180.4 ng/m l ). Conclusions: Although platelet aggregation did not increase, there was an increase in serum COX-metabolized eicosanoid mediators such as TxB₂ in healthy medical staff after night-shift work. This platelet hypersensitivity may be one of the mechanisms underlying the significant association between night-shift work and adverse cardiovascular outcomes.