The dietary intake of n-3 (omega-3) long-chain PUFAs (LC-PUFAs) from fish may improve endothelial function and arterial stiffness. The objective was to test the hypothesis that increasing intakes of n-3 LC-PUFAs-equivalent to the consumption of 1, 2, or 4 portions of oily fish per week-favorably affects endothelial function and arterial stiffness. A parallel-design, randomized, double-blind study compared daily doses of 0.45, 0.9, and 1.8 g n-3 LC-PUFAs (EPA:DHA ratio of 1.51:1) with placebo (refined olive oil). The primary and secondary outcomes were changes in flow-mediated dilatation (FMD) of the brachial artery, arterial stiffness, and blood pressure. Nonsmoking men (n = 142) and women (n = 225) aged 45-70 y were randomly assigned to treatment for 12 mo; 312 subjects completed the intervention. Compliance with the intervention was corroborated by significant dose-dependent increases in the proportions of EPA and DHA in erythrocyte lipids and a 16.5% reduction in serum triacylglycerol concentrations with 1.8 g n-3 LC-PUFAs/d. FMD was lower in men than in women (P < 0.0001) and decreased with age (ρ = 0.270, P < 0.001) but was not significantly (P = 0.781) related to n-3 LC-PUFA intake. The mean changes in FMD (95% CIs) compared with placebo were 0.1% (-0.9%, 1.1%), -0.3% (-1.3%, 0.6%), and -0.3% (-1.3%, 0.7%) with daily intakes of 0.45, 0.9, and 1.8 g n-3 LC-PUFAs, respectively. No significant treatment effects were noted for arterial stiffness and central mean or 24-h ambulatory blood pressure. Intakes of n-3 LC-PUFAs ≤1.8 g/d do not improve endothelial function in healthy adults. The trial is registered at controlled-trials.com as ISRCTN66664610.