Aortic stiffness, determined by carotid-femoral pulse wave velocity (cfPWV), independently predicts cardiovascular outcomes. Recent studies suggest that glucose levels influence arterial stiffness indices. It is not clear, however, whether glucose affects cfPWV independently of glucoregulatory hormones. The aim of this study was to utilize a pancreatic clamping approach to determine whether plasma glucose independently predicts cfPWV. Healthy participants (N = 10) underwent pancreatic clamping to control glucose at varying concentrations using a 20% dextrose infusion while suppressing endogenous glucagon, insulin, and growth hormone by octreotide and replacing the hormones intravenously to achieve basal concentrations. Tonometric cfPWV, blood pressure, heart rate, plasma glucose, glucagon, insulin, growth hormone, and vasoactive biomarkers were measured. Plasma glucose levels of 150 mg/dl at 1 hr and 200 mg/dl at 2 hr postbaseline were achieved. There were no significant changes in cfPWV (5.8 m/s at 0 hr, 5.9 m/s at 1 hr, and 5.9 m/s at 2 hr) with increased glucose levels. There were small increases in insulin secretion. A definitive role for glucose in cfPWV modulation was not determined; there is a potential role for insulin as a cfPWV modulator. Continued efforts in clarifying the independent roles of glucose and insulin can elucidate novel vessel-related targets for cardiovascular disease prevention and management in patients with impaired glucose tolerance and diabetes.