Venous occlusion plethysmography (VOP) is a valid non-invasive method to assess peripheral blood flow (BF) in humans. Our aim was to determine intra- and inter-tester reproducibility of BF analysis using a traditional manual method and a novel system, based on a semi-automatic approach. Ten healthy subjects and ten subjects with chronic heart failure (CHF) were evaluated. Blood flow was measured on the forearm at baseline and after 5 min of circulatory occlusion (reactive hyperemia (RH)). Two testers independently and blindly analyzed each VOP recording. Both methods were highly reproducible intra- and inter-testers. In addition, there was a high association between the methods, since the intraclass correlation coefficients (ICCs) for healthy subjects were 0.99, 0.99 and 0.99, and the coefficients of variation (CVs) were 1.8, 2.4 and 1.6% for baseline, RH peak and RH area under the curve, respectively. For CHF subjects, the ICCs were 0.99, 0.98 and 0.99, and the CVs were 2.9, 3.6 and 2.0%. In addition, the time spent on the semi-automatic analyses was shorter (p < 0.05). In conclusion, both methods demonstrated high intra- and inter-tester reproducibility for baseline and RH BF analysis. However, since the semi-automatic method was faster to generate the results, the present study supports its usage for the analysis of BF measured by VOP.