Flow-mediated dilation (FMD) is a widely used tool to investigate endothelial function. However, FMD assessment may cause mechanical damage to the arterial endothelium. In this study we investigated the effect of FMD assessment on endothelial function. We studied 20 healthy subjects (26 ± 6 years; 12 males). FMD was assessed by measuring brachial artery dilation in response to hyperemia after 5 min of forearm cuff inflation. Subjects were studied on 2 subsequent days. On day 1 they underwent two consecutive FMD measures, with the second test (FMD2) performed 15 min after the first test (FMD1). On day 2, the subjects were randomized to receive either placebo (saline) or intravenous l-arginine (10 g in 20 min). At the end of the infusion, patients underwent two consecutive FMD measures following the same protocol as on day 1. Asymmetric dimethyl-arginine (ADMA) serum levels were assessed on day 2 before FMD1 and FMD2. On day 1, FMD2 was lower than FMD1 in both groups (placebo 6.47 ± 2.1 vs. 7.86 ± 1.8%, P < 0.01; arginine 6.13 ± 2.6 vs. 7.76 ± 2.7%, P < 0.01). On day 2, a significant reduction of FMD was observed during FMD2 compared to FMD1 in the placebo group (5.82 ± 1.7 vs. 7.44 ± 2.2%, P < 0.001), but not in the arginine group (7.19 ± 1.5 vs. 7.27 ± 1.5, P = 0.67). ADMA levels significantly increased compared to baseline after FMD1 (0.59 ± 0.12–0.91 ± 0.64 μmol/l, P = 0.036), with similar changes in the two groups. FMD assessment induces a significant impairment of endothelial function. An increase of endogenous NO synthesis inhibitors seems responsible for the phenomenon that is reversed by l-arginine administration.