The effect of acute hyperglycemia per se on coronary perfusion in humans is undefined. We evaluated the effects of short-term hyperglycemia on myocardial blood flow reserve (MBFR) in healthy nondiabetic volunteers. Twenty-one nondiabetic volunteers (76 % females, mean ± SD, age 48 ± 5 years) had noninvasive MBFR assessment while exposed to pancreatic clamp with somatostatin and replacement glucagon and growth hormone infusions, with frequent interval plasma glucose (PG) monitoring. Insulin was infused at 0.75 mU/kg/min to mimic postprandial plasma insulin concentrations, and glucose was infused to maintain euglycemia (PG 93.9 ± 7.3 mg/dl) followed by hyperglycemia (PG 231.5 ± 18.1 mg/dl). Myocardial contrast echocardiography (MCE) was performed during each glycemic steady state using continuous infusion of Definity at rest and during regadenoson (Lexiscan 5 ml (400 μg) intravenous bolus) infusion to quantify myocardial blood flow (MBF) and determine MBFR. Insulin resistance (IR) was assessed by glucose infusion rate (GIR; mg/kg/min) at euglycemia. Median stress MBF, MBFR, and β reserve were significantly reduced during acute hyperglycemia versus euglycemia (stress MBF 3.9 vs 5.4, P = 0.02; MBFR 2.0 vs 2.7, P < 0.0001; β reserve 1.45 vs 2.4, P = 0.007). Using a median threshold GIR of 5 mg/kg/min, there was a correlation between GIR and hyperglycemic MBFR (r = 0.506, P = 0.019). MBFR, as determined noninvasively by MCE, is significantly decreased during acute hyperglycemia in nondiabetic volunteers, and the magnitude of this reduction is modulated by IR.