Aim: Endothelial dysfunction is the earliest change in atherosclerosis. Flow-mediated vasodilatation (FMD) is used to assess endothelial function in humans. However, it is not easy to do this assessment in small animals. We designed an instrument that uses infrared sensors to assess FMD in vivo using a rodent model. Methods: Twenty-four adult male Wistar Kyoto (WKY) rats were randomly divided into three groups. The animals were anesthetized with intraperitoneal injection of urethane (1.2 mg/kg; Sigma-Aldrich, St. Louis, MO, USA). To stabilize body temperature, each animal was placed on a heated electric blanket with a warming lamp overhead. FMD was measured under continuous infusion of normal saline followed by intra-arterial infusion of either acetylcholine (Ach; n=8), sodium nitroprusside (SNP; n=8), or Nω-Nitro-L-arginine methyl ester (L-NAME; n=8). Results: The dilatation indices (DIs) of all three groups were similar before the application of the vasoactive agents, (1.82 ± 0.46, 1.81 ± 0.44, and 1.91 ± 0.40, p=0.877, by one-way ANOVA). The DI was significantly increased during infusion of Ach (2.97 ± 1.03 vs. 1.82 ± 0.46, p=0.015), unchanged during the infusion of SNP (1.81 ± 0.44 vs. 1.98 ± 0.40, p=0.574), and attenuated during the infusion of L -NAME (1.91 ± 0.40 vs. 1.42 ± 0.35; p =0.028). Conclusions: The results of the present study correlated well with those of previous human studies, suggesting that the method can be used for in vivo evaluation of endothelial function in small animals.