ScopeLack of biomarkers is a challenge for the accurate assessment of protein intake and interpretation of observational study data. The study aims to identify biomarkers of a protein‐rich dietary pattern.Methods and ResultsThe Optimal Macronutrient Intake Trial to Prevent Heart Disease (OmniHeart) trial is a randomized cross‐over feeding study which tested three dietary patterns with varied macronutrient content (carbohydrate‐rich; protein‐rich with about half from plant sources; and unsaturated fat‐rich). In 156 adults, differences in log‐transformed plasma metabolite levels at the end of the protein‐ and carbohydrate‐rich diet periods using paired t‐tests is examined. Partial least‐squares discriminant analysis is used to identify a set of metabolites which are influential in discriminating between the protein‐rich versus carbohydrate‐rich dietary patterns. Of 839 known metabolites, 102 metabolites differ significantly between the protein‐rich and the carbohydrate‐rich dietary patterns after Bonferroni correction, the majority of which are lipids (n = 35), amino acids (n = 27), and xenobiotics (n = 24). Metabolites which are the most influential in discriminating between the protein‐rich and the carbohydrate‐rich dietary patterns represent plant protein intake, food or beverage intake, and preparation methods.ConclusionsThe study identifies many plasma metabolites associated with the protein‐rich dietary pattern. If replicated, these metabolites may be used to assess level of adherence to a similar dietary pattern.