Background: Evaluation of the dietary fat consumption in athletes following vegan diets is scarce. The aim of this study was to explore the intakes, availability, and uptake of physiologically relevant fatty acids into whole blood, and consequently the Omega-3 Index (O3I) of endurance athletes following vegan and omnivorous dietary patterns. Materials: Males aged 18 to 55 years, engaging in ≥ four hours of training/week and following a vegan (>6 months) or omnivorous dietary pattern were eligible to participate. A 7-day food and training diary was collected and an incremental ramp running protocol used to determine peak aerobic capacity. A finger prick blood sample was collected to determine the whole blood fatty acid profile and O3I. Participants were grouped as following a vegan or omnivorous diet matched for age, training volume and peak aerobic capacity. Results: The vegan group (n = 12) consumed significantly less dietary total fat (122.2 g/day vs 84.1 g/day p = 0.007), saturated fat (43.74 g/day vs 18.42 g/day p < 0.0001), monounsaturated fat (49.6 g/day vs 35.64 g/day p = 0.039) and long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) compared to the omnivorous group (n = 8). Between group differences in whole blood fatty acid concentrations were observed including; linoleic, eicosapentaenoic, docosahexaenoic, n-6:n-3 and AA:EPA ratios. O3I in both groups were suboptimal (vegan: 4.13%, omnivorous: 5.40%) in terms of cardiac risk. Conclusion: Male endurance athletes should ensure their dietary LC n-3 PUFA intakes, particularly EPA and DHA fatty acids are sufficient to optimize their O3I.