Long-chain polyunsaturated fatty acids (LC-PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary LC-PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. Female C57BL/6J mice consumed diets containing increasing amounts of fish oil (FO) ad libitum for 8 weeks. RBC deformability, filterability, and post-transfusion recovery (PTR) were evaluated before and after cold storage. Lipidomics and lipid peroxidation markers were evaluated in fresh and stored RBCs. High-dose dietary FO (50%, 100%) was associated with a reduction in RBC quality (i.e., in vivo lifespan, deformability, lipid peroxidation) along with a reduced 24 h PTR after cold storage. Low-dose dietary FO (6.25–12.5%) improved the filterability of fresh RBCs and reduced the lipid peroxidation of cold-stored RBCs. Although low doses of FO improved RBC deformability and reduced oxidative stress, no improvement was observed for the PTR of stored RBCs. The improvement in RBC deformability observed with low-dose FO supplementation could potentially benefit endurance athletes and patients with conditions resulting from reduced perfusion, such as peripheral vascular disease.