Objectives: To determine the effects of n-3 polyunsaturated fatty acid (PUFA) supplementation, i.e., α-linolenic (~230 mg), eicosapentaenoic (~15 mg), and docosahexaenoic acid (~105 mg), on the prevalence of T helper (Th17) and regulatory (Treg) lymphocytes, cytokine profile, other inflammatory parameters and lipid metabolites, microvascular reactivity and endothelium-dependent/-independent mechanisms of vasodilation in healthy individuals. Study Design and Setting: This was a randomized, double-blind, placebo-controlled, prospective, intervention study. Research was carried out at Department of Physiology and Immunology, Faculty of Medicine Osijek, Josip Juraj Strossmayer University of Osijek in Osijek, Croatia Participants and Methods: 44 healthy adults of both sexes were recruited according to inclusion/exclusion criteria for the participation in the study. 42 participants were randomized and divided into: 1) Control group (N=21) that consumed regular hen eggs (n-3 PUFAs: ~249 mg/per day), 2) n-3 PUFAs group (N=21) that consumed enriched hen eggs (n-3 PUFAs: ~1053 mg/per day). Two participants from n-3 PUFA were lost to follow-up due to personal reasons, with total number of participants included in final analysis being N=19. The study protocol included two appointments and participants were instructed to eat three hard-boiled hen eggs/day for three weeks. Venous blood samples were taken before and after the dietary protocol for serum and peripheral blood mononuclear cells collection/isolation. Anthropometric and biochemical measurements, blood pressure, body composition and fluid status, serum lipid and free fatty acids profile were measured before and after respective dietary protocol. Concentrations of lipid mediators, inflammatory and endothelial activation markers were measured via ELISA and Luminex assays. Frequencies of Tregs and Th17 lymphocytes were determined by flow cytometry. Skin microvascular blood flow in response to iontophoresis of acetylcholine and sodium nitroprusside was assessed by laser Doppler 8. SUMMARY 71 flowmetry. An adjusted dietary assessment questionnaire was used to determine the dietary habits of the study population. Results: Both study groups showed reduced frequencies of peripheral Treg and Th17 lymphocytes. Functional capacity for cytokine secretion was significantly altered in the n-3 PUFAs group through increased anti- (transforming growth factor β1, interleukin 10) and reduced pro-inflammatory (interleukin 6, interferon gamma) cytokine secretion. Significantly increased levels of resolvins were observed only in the n-3 PUFAs group as well as significantly improved acetylcholine-induced dilatation. Conclusion: Diet supplemented with n-3 PUFAs alters immune response towards inflammation resolving conditions through effects on lipid mediators and cytokine secretion by T lymphocytes, as well as it provides potential mechanisms for enhanced microvascular function since it improved microvascular endothelium-dependent vasodilation in human model without underlying comorbidities. Trial registration: Clinical Trials ID NCT02720250