OBJECTIVES: Endothelial function can be assessed by acetylcholine (ACh) iontophoresis with single current application. The effect of inter-electrode distance as well as electrical cutaneous resistance (ECR) on ACh dependent vasodilation has never been studied using single current application. The aims of this study are (i) to compare ACh-peak and ECR measured at different inter-electrode distances, (ii) to assess the relationship between ACh-peak and ECR, (iii) and to study the reproducibility of the ECR values. METHODS: Fourteen healthy subjects were included. Using laser speckle contrast imaging, ACh-iontophoreses (0.1 mA, 30s) were performed on the forearm at a 7-day interval with an inter-electrode distance set at 5 cm. Two other inter-electrode distances were also evaluated: 10 cm and 15 cm. ECR was measured during each ACh-iontophoresis as well as the ACh-peak. RESULTS: No statistical difference was found between the ACh-peak values obtained at 5 cm, 10 cm and 15 cm. ECRs were also not statistically different. An inverse relationship (r=-0.60) was found between the ACh-peak and ECR (p\textless0.05). The coefficient of variation of the inter-day reproducibility of the ECR values was 9.1% [6.5%-15.1%] with an intra-class-correlation coefficient of 0.93 [0.81-0.98]. CONCLUSION: Inter-electrode distance ranging from 5 cm to 15 cm changes neither the ACh-peak value nor the ECR value. ECR impacts ACh-peak values.