BACKGROUND: Exposure to air pollution is associated with elevated cardiovascular risk. Evidence shows that omega-3 polyunsaturated fatty acids (omega-3 PUFA) may attenuate the adverse cardiovascular effects of exposure to fine particulate matter (PM2.5). However, it is unclear whether habitual dietary intake of omega-3 PUFA protects against the cardiovascular effects of short-term exposure to low-level ambient air pollution in healthy participants. In the present study, sixty-two adults with low or high dietary omega-3 PUFA intake were enrolled. Blood lipids, markers of vascular inflammation, coagulation and fibrinolysis, and heart rate variability (HRV) and repolarization were repeatedly assessed in 5 sessions separated by at least 7 days. This study was carried out in the Research Triangle area of North Carolina, USA between October 2016 and September 2019. Daily PM2.5 and maximum 8-h ozone (O3) concentrations were obtained from nearby air quality monitoring stations. Linear mixed-effects models were used to assess the associations between air pollutant concentrations and cardiovascular responses stratified by the omega-3 intake levels. RESULTS: The average concentrations of ambient PM2.5 and O3 were well below the U.S. National Ambient Air Quality Standards during the study period. Significant associations between exposure to PM2.5 and changes in total cholesterol, von Willebrand factor (vWF), tissue plasminogen activator, D-dimer, and very-low frequency HRV were observed in the low omega-3 group, but not in the high group. Similarly, O3-associated adverse changes in cardiovascular biomarkers (total cholesterol, high-density lipoprotein, serum amyloid A, soluable intracellular adhesion molecule 1, and vWF) were mainly observed in the low omega-3 group. Lag-time-dependent biphasic changes were observed for some biomarkers. CONCLUSIONS: This study demonstrates associations between short-term exposure to PM2.5 and O3, at concentrations below regulatory standard, and subclinical cardiovascular responses, and that dietary omega-3 PUFA consumption may provide protection against such cardiovascular effects in healthy adults.