Abstract Objectives The aim of our study was to assess whether acute, short-term hyperglycemia affects platelet reactivity in patients with Type II diabetes mellitus (T2DM). Background Hyperglycemic spikes are thought to precipitate ischemic events in T2DM. Previous studies have shown in vivo platelet activation in diabetes; however, no studies have assessed whether acute in vivo hyperglycemia induces further activation of platelets. Methods In a cross-over, randomized, double-blind study, 12 patients with T2DM underwent 4 h of either acute hyperglycemia (13.9 mmol/l, 250 mg/dl) or euglycemia (5.5 mmol/l, 100 mg/dl). Shear stress-induced platelet activation, P-selectin and lysosomal integral membrane protein (LIMP) expression on platelets in the bleeding-time blood, urinary 11-dehydro-thromboxane B 2 (TxB 2) excretion, von Willebrand factor:antigen (vWF:Ag), and von Willebrand factor:activity (vWF:activity) were measured before and after hyperglycemia or euglycemia. Results Shear stress-induced platelet activation, P-selectin and LIMP expression on platelets in the bleeding-time blood, and urinary 11-dehydro-TxB 2 excretion increased significantly after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. Plasma vWF:Ag and vWF:activity increased strikingly in parallel fashion after hyperglycemic clamping, whereas no changes were observed after euglycemic clamping. Conclusions Our data demonstrate that acute, short-term hyperglycemia induces an increased activation of platelets exposed to high shear stress conditions in vitro (filtration method) or in vivo (bleeding time). In vivo platelet activation is reflected by an increased urinary excretion of 11-dehydro-TxB 2. The increased levels of vWF in the circulation correlate with the increase in platelet activation markers and may indicate some degree of causation. Acute, short-term hyperglycemia in T2DM may precipitate vascular occlusions by facilitating platelet activation.