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Dietary omega-3 polyunsaturated fatty acids (PUFAs) are readily incorporated into heart and skeletal muscle membranes where, in the heart, animal studies show they reduce O2 consumption. To test the hypothesis that omega-3 PUFAs alter O2 efficiency in humans, the effects of fish oil (FO) supplementation on O2 consumption during exercise were evaluated. Sixteen well-trained men (cyclists), randomly assigned to receive 8 × 1 g capsules per day of olive oil (control) or FO for 8 weeks in a double-blind, parallel design, completed the study (control: n = 7, age 27.1 ± 2.7 years; FO: n = 9, age 23.2 ± 1.2 years). Subjects used an electronically braked cycle ergometer to complete peak O2 consumption tests (VO2peak) and sustained submaximal exercise tests at 55% of peak workload (from the VO2peak test) before and after supplementation. Whole-body O2 consumption and indirect measurements of myocardial O2 consumption [heart rate and rate pressure product (RPP)] were assessed. FO supplementation increased omega-3 PUFA content of erythrocyte cell membranes. There were no differences in VO2peak (mL kg−1 min−1) (control: pre 66.8 ± 2.4, post 67.2 ± 2.3; FO: pre 68.3 ± 1.4, post 67.2 ± 1.2) or peak workload after supplementation. The FO supplementation lowered heart rate (including peak heart rate) during incremental workloads to exhaustion (P < 0.05). In addition, the FO supplementation lowered steady-state submaximal exercise heart rate, whole-body O2 consumption, and RPP (P < 0.01). Time to voluntary fatigue was not altered by FO supplementation. This study indicates that FOs may act within the healthy heart and skeletal muscle to reduce both whole-body and myocardial O2 demand during exercise, without a decrement in performance.