Introduction: Over time, diabetes damages both the micro- and macrovasculature. Acutely, hyperglycemia causes oxidative stress and multiple biochemical abnormalities in endothelial cells. However, initial in vivo functional vascular responses to acute hyperglycemia (AH) are not well-characterized in humans or animals. Measurement of microvascular perfusion (MP) and endothelial function provides in vivo estimates of nutrient and hormone access to tissues and endothelial health, respectively. Methods: Thirteen healthy adults (18-35 years, BMI 18-25 kg/m2) were fasted overnight. Octreotide was infused with basal insulin replacement and baseline measures of endothelial function by flow-mediated dilation (FMD) and MP (heart and skeletal muscle) by contrast-enhanced ultrasound were obtained after 90 minutes. Glucose was infused to maintain fasting euglycemia (EU) or raise glucose to ∼200 mg/dL (AH) for 4 hours with measures of FMD and MP repeated. Linear mixed model was used for statistical analysis, with Bonferroni correction for between-study comparisons. Results: Compared to baseline, 4 hours of AH significantly increased skeletal muscle microvascular blood volume (MBV) (p=0.0002), microvascular flow velocity (p=0.047), and microvascular blood flow (p=0.025). MBV significantly increased in comparison to euglycemia (p=0.02). MBV also increased in cardiac muscle during AH (p=0.002). FMD trended towards a significant increase with AH (p=0.058). No changes were observed with EU. Discussion: First, octreotide had no discernible effect on MP or FMD, which allowed testing the effect of AH while insulin remained at basal. Second, we provide the first evidence that heart and skeletal muscle respond to 4 hours of AH by increasing perfusion in healthy subjects. Whether this response is maintained with chronic hyperglycemia is unknown. Disclosure W.B. Horton: None. L. Jahn: None. L. Hartline: None. J.T. Patrie: None. E. Barrett: Research Support; Self; AstraZeneca. Funding National Institutes of Health (F32HL142304-01)