Some studies suggest that mean platelet volume (MPV) correlates with increased risk for cardiovascular morbidity and mortality. In this study, we aim to assess reproducibility, need for standardized measurements, effect of aspirin, and association with other established markers of platelet activity. Following an overnight fast, 48 healthy volunteers had weekly assessment of platelet activity and were administered aspirin 81 mg daily for 7 d between weeks 3 and 4. We investigated the influence of time between phlebotomy and MPV measurement (n = 10). Reproducibility was assessed by coefficient of variation (CV) and intraclass correlation coefficient (ICC). MPV measurements were reproducible (Week 1: 10.6 fL [9.9–11], Week 2: 10.6 fL [10.0–10.9], Week 3: 10.6 fL [9.8–11]). CV was ≤4% and ICC > 0.85 (p < 0.001) for each comparison, indicating excellent reproducibility. There was no effect of aspirin on MPV (10.6 fL [9.8–11] versus 10.5 fL [9.9–11]; p = 0.81). MPV significantly increased as time between phlebotomy and MPV measurement increased (Spearman’s rho = 0.94, p = 0.001). Increasing MPV tertiles was associated with collagen- and thrombin receptor-activated peptide-induced platelet aggregation but not with ADP- or arachidonic acid-induced or spontaneous platelet aggregation. In conclusion, when standardized, MPV is a reproducible marker of platelet size and not affected by low-dose aspirin. MPV is modestly associated with some, but not all, markers of platelet activity.