The purpose of this investigation was to determine the independent and combined effects of aerobic exercise and omega-3 fatty acid (n-3fa) supplementation on lipid and lipoproteins. Sedentary, normoglycemic, nonsmoking men (n = 11) were assigned to perform rest and exercise before and during n-3fa supplementation. Exercise consisted of 3 consecutive days of treadmill walking at 65% maximum O(2) consumption for 60 min. Supplementation consisted of 42 days of 4.55 g/day of n-3fa. A two-way factorial ANOVA with repeated measures revealed significant reductions in total cholesterol (P = 0.001, -9.2%) and triglyceride (P = 0.007, -32.4%) concentrations postexercise. In addition, exercise increased LDL peak particle size (P = 0.001) from 26.2 to 26.4 nm, but not HDL size. The n-3fa supplementation resulted in a significant shift in the distribution of HDL-cholesterol (HDL-C) carried by HDL(2b+2a) (P = 0.001, 14.2%) and HDL(3a+3b) (P = 0.001, -22.8%), despite no significant changes in lipid and lipoprotein-cholesterol concentrations. The majority of the shift in HDL-C was noted in HDL(2b) (P = 0.001, 20.9%) and HDL(3a) (P < 0.001, -31.0%) particles. There were no combined effects of exercise and n-3fa supplementation on lipids and lipoproteins. Three consecutive days of aerobic exercise reduced triglyceride and total cholesterol concentrations with a concomitant increase in LDL peak particle size. In contrast, n-3fa supplementation shifted HDL-C from HDL(3) particles to HDL(2) particles, despite no significant changes in HDL(2)-C and HDL(3)-C concentrations. Exercise and n-3fa supplementation do not synergistically improve serum lipids and lipoproteins, but rather independently affect the metabolism of lipids and lipoproteins.