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Scope: Fish intake is reported to be associated with certain health benefits; however, accurate assessment of fish intake is still problematic. The objective of this study is to identify fish intake biomarkers and examine relationships with health parameters in a free-living population., Methods and Results: In the NutriTech study, ten participants randomized into the fish group consume increasing quantities of fish for 3 days per week for 3 weeks. Urine is analyzed by NMR spectroscopy. Trimethylamine-N-oxide (TMAO), dimethylamine, and dimethyl sulfone are identified and display significant dose-response with intake (p < 0.05). Fish consumption yields a greater increase in urinary TMAO compared to red meat. Biomarker-derived fish intake is calculated in the National Adult Nutrition Survey cross-sectional study. However, the correlation between fish intake and TMAO (r = 0.148, p < 0.01) and that between fish intake and calculated fish intake (r = 0.142, p < 0.01) are poor. In addition, TMAO shows significantly positive correlation with serum insulin and insulin resistance in males and the relationship is more pronounced for males with high dietary fat intake., Conclusion: Urinary TMAO displays a strong dose-response relationship with fish intake; however, use of TMAO alone is insufficient to determine fish intake in a free-living population., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)