BackgroundDietary consumption has traditionally been studied through food intake questionnaires. Metabolomics can be used to identify blood markers of dietary protein that may complement existing dietary assessment tools.ObjectivesWe aimed to identify associations between 3 dietary protein sources (total protein, animal protein, and plant protein) and serum metabolites using data from the Atherosclerosis Risk in Communities Study.MethodsParticipants’ dietary protein intake was derived from a food frequency questionnaire administered by an interviewer, and fasting serum samples were collected at study visit 1 (1987–1989). Untargeted metabolomic profiling was performed in 2 subgroups (subgroup 1:n= 1842; subgroup 2:n= 2072). Multivariable linear regression models were used to assess associations between 3 dietary protein sources and 360 metabolites, adjusting for demographic factors and other participant characteristics. Analyses were performed separately within each subgroup and meta-analyzed with fixed-effects models.ResultsIn this study of 3914 middle-aged adults, the mean (SD) age was 54 (6) y, 60% were women, and 61% were Black. We identified 41 metabolites significantly associated with dietary protein intake. Twenty-six metabolite associations overlapped between total protein and animal protein, such as pyroglutamine, creatine, 3-methylhistidine, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. Plant protein was uniquely associated with 11 metabolites, such as tryptophan betaine, 4-vinylphenol sulfate,N-δ-acetylornithine, and pipecolate.ConclusionsThe results of 17 of the 41 metabolites (41%) were consistent with those of previous nutritional metabolomic studies and specific protein-rich food items. We discovered 24 metabolites that had not been previously associated with dietary protein intake. These results enhance the validity of candidate markers of dietary protein intake and introduce novel metabolomic markers of dietary protein intake.