Background: The sum of eicosapentaenoic acid (EPA, 20:5 ω3) and docosahexaenoic acid (DHA, 22:6 ω3) in erythrocyte membranes, termed the omega-3 index, can indicate suboptimal intake of omega-3 fatty acids, a risk factor for cardiovascular disease (CVD). To study the effects of fatty acid supplementation, we investigated the rate of incorporation and clearance of these fatty acids in erythrocyte membranes and plasma after intake of supplements. Methods: Twenty study participants received supplementation with either fish oil (1296 mg EPA + 864 mg DHA/day) or flaxseed oil (3510 mg alpha-linolenic acid + 900 mg linoleic acid/day) for 8 weeks. We obtained erythrocyte membrane and plasma samples at weeks 0, 4, 8, 10, 12, 14, 16, and 24 and extracted and analyzed fatty acids by gas chromatography. Results: After 8 weeks of fish oil supplementation, erythrocyte membrane EPA and DHA increased 300% (P <0.001) and 42% (P <0.001), respectively. The mean erythrocyte omega-3 index reached a near optimal value of 7.8%, and remained relatively high until week 12. EPA and DHA showed greater increases and more rapid washout period decreases in plasma phospholipids than in erythrocyte membranes. Flaxseed oil supplementation increased erythrocyte membrane EPA to 133% (P <0.05) and docosapentaenoic acid (DPA, 22:5 ω3) to 120% (P <0.01) of baseline, but DHA was unchanged. In plasma phospholipids, EPA, DPA, and DHA showed a slight but statistically insignificant increase. Conclusions: Erythrocyte membrane EPA+DHA increases during relatively short intervals in response to supplementation at rates related to amount of supplementation. These results may be useful to establish appropriate dosage for omega-3 fatty acid supplementation.