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<h4>Background</h4>Effect of fish oil supplementation on flow-mediated dilation, an index of endothelial function in humans, remains controversial. We performed a meta-analysis to determine whether fish oil supplementation could improve endothelial function.<h4>Methods</h4>Human intervention studies were identified by systematic searches of Medline, Embase, Cochrane's library and references of related reviews and studies. A random-effect model was applied to estimate the pooled results. Meta-regression and subgroup analyses were performed to evaluate the impact of study characteristics on the effect of fish oil supplementation on flow-mediated dilation.<h4>Results</h4>A total of sixteen records with 1,385 subjects were reviewed. The results of the pooled analysis showed that fish oil supplementation significantly improved flow-mediated dilation (weighed mean difference: 1.49%, 95% confidence interval 0.48% to 2.50%, p = 0.004). Meta-regression and subgroup analysis suggested that the quality of included studies were inversely related to the overall effect (regression coefficient  = -1.60, p = 0.04), and the significance of the effect was mainly driven by the studies with relatively poor quality. Sensitivity analysis including only double-blind, placebo-controlled studies indicated fish oil supplementation has no significant effect on endothelial function (weighed mean difference: 0.54%, 95% confidence interval -0.25% to 1.33%, p = 0.18). Besides, normoglycemic subjects or participants with lower diastolic blood pressure seemed to be associated with remarkable improvement of endothelial function after fish oil supplementation.<h4>Conclusions</h4>Although current evidence suggested a possible role of fish oil in improving endothelial function, large-scale and high-quality clinical trials are needed to evaluate these effects before we can come to a definite conclusion.