Introduction: Iontophoresis of vasoactive substances such as acetylcholine (ACh) and sodium nitroprusside (SNP) combined with Laser Doppler fluximetry (LDF) is a non-invasive tool used to determine microvascular endothelial function. This study aims to test the effect of sodium chloride on non-specific vasodilatation when used as a vehicle in the process of iontophoresis. This study also aims to define the number of current pulses needed to get the maximum effect during iontophoresis with ACh and SNP using low current strength. Methods: The experiment was conducted in five healthy females. Baseline skin perfusion was taken before administration of seven current pulses. Current strength of 0.007 mA and current density of 0.01 mA/cm2 were used. Acetylcholine was used to assess endothelial dependent vasodilatation, while SNP was used to assess endothelial independent vasodilatation. The mean skin perfusion (AU) responses to the iontophoresis of ACh at the anodal and SNP at the cathode leads were recorded. Sodium chloride (0.9%) was used as a vehicle to obtain concentration of 1% for both ACh and SNP. Iontophoresis of pure vehicle (NaCl) was conducted on a separate day to observe the effect of vehicle only on the iontophoresis process at both anode and cathode. Results: Iontophoresis of NaCl showed no significant increase in perfusion compared to baseline at both anode and cathode. Significant increases in skin perfusion were observed with SNP and ACh; a plateau of ACh was reached from the 3rd pulse onwards; while the plateau of SNP was reached from the 4th pulse onwards. Conclusion: NaCl could be used as a vehicle for Ach and SNP during iontophoresis as it did not cause non-specific vasodilatation. Using five current pulses are adequate for iontophoresis of ACh and SNP to assess microvascular endothelial function.