OBJECTIVES: To assess whether the validity of endothelial function measurement by flow-mediated dilatation (FMD) is affected by local brachial artery stiffness (distensibility coefficient; DC) and arterial wall thickness (intima-media thickness, IMT). BACKGROUND: FMD measurement relies on assessment of arterial diameter change. Increased IMT and decreased DC might physically limit dilatation of the brachial artery in spite of healthy endothelium. METHODS: DC, IMT and FMD of the brachial artery were simultaneously measured in 349 patients with advanced atherosclerosis or cardiovascular risk factors. The relations between FMD and age, and FMD and current smoking were regarded as a proxy for the relation between FMD and true endothelial function. RESULTS: The relations between FMD and age, and FMD and smoking, were significantly modified by brachial artery DC. No modification was found for IMT. The interaction terms were statistically significant (p=0.03 and 0.04, respectively). The relation between FMD and age, and FMD and smoking was progressively more pronounced in patients with more elastic arteries. CONCLUSION: The results of our study indicate that increased arterial stiffness may interfere with valid measurement of FMD and that patients with stiff arteries may be considered for exclusion from analyses involving FMD to ensure its validity.