Post-occlusive reactive hyperaemia (PORH) and vasodilation induced by acetylcholine (ACh) iontophoresis are tests of endothelial function that can be studied with laser speckle contrast imaging (LSCI). LSCI has the advantage of having good temporal and spatial resolutions but can lead to a high amount of data when several minutes of recordings are needed. Parameters of PORH and ACh iontophoresis vasodilation are therefore often determined by several observers or by the same observer on different days. Nevertheless, inter- and intra-observer reproducibility for the determination of such parameters has not been studied yet. We analyzed inter-observer and intra-observer reproducibility of baseline, peak and plateau determination for the two microvascular tests. Ten recordings of both PORH and ACh iontophoresis have been analyzed by two blinded trained observers. For peak determination, inter-observer coefficient of variation (CV) was 4.7% and 3.0% for PORH and ACh respectively. Intra-observer reproducibility expressed in CV ranges from 2.4% to 5.4% for PORH-peak and ACh-peak. CVs for peak determination are better than for baseline or plateau determination for both microvascular tests. This suggests that when microvascular vasodilations are reported, the data segments measured have to be noted. Finally microvascular tests using LSCI have an excellent intra- and inter-observer reproducibility.