Reduced peripheral microvascular reactivity is associated with increased risk for major adverse cardiac events (MACE). Tools for non-invasive assessment of peripheral microvascular function are limited and existing technology is poorly validated in both healthy populations and patients with cardiovascular disease (CVD). Here, we utilized a handheld incident-darkfield imaging tool (CytoCam) to test the hypothesis that compared to healthy individuals (no risk factors for CVD), subjects formally diagnosed with coronary artery disease (CAD) or those with ≥ 2 risk factors for CAD (Endothelial Dysfunction; ED) would exhibit impaired peripheral microvascular reactivity. A total of 17 participants (11 healthy, 6 ED) were included in this pilot study. CytoCam was used to measure sublingual microvascular total vessel density (TVD), perfused vessel density (PVD), and microvascular flow index (MFI) in response to topical application of acetylcholine (Ach) and sublingual administration of nitroglycerin (NTG). Baseline MFI and PVD were significantly reduced in the ED cohort compared to healthy individuals. Surprisingly, following application of acetylcholine and nitroglycerin, both groups showed a significant improvement in all 3 microvascular perfusion parameters. These results suggest that despite baseline reductions in both microvascular density and perfusion, human in vivo peripheral microvascular reactivity to both endothelial-dependent and -independent vasoactive agents remains intact in individuals with CAD or multiple risk factors for disease.