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An altered amino acid metabolism has been described in frail older adults which may contribute to muscle loss and functional decline associated with frailty. In the present investigation, we compared circulating amino acid profiles of older adults with physical frailty and sarcopenia (PF&S, <i>n</i> = 94), frail/pre-frail older adults with type 2 diabetes mellitus (F-T2DM, <i>n</i> = 66), and robust non-diabetic controls (<i>n</i> = 40). Partial least squares discriminant analysis (PLS–DA) models were built to define the amino acid signatures associated with the different frailty phenotypes. PLS–DA allowed correct classification of participants with 78.2 ± 1.9% accuracy. Older adults with F-T2DM showed an amino acid profile characterized by higher levels of 3-methylhistidine, alanine, arginine, ethanolamine, and glutamic acid. PF&S and control participants were discriminated based on serum concentrations of aminoadipic acid, aspartate, citrulline, cystine, taurine, and tryptophan. These findings suggest that different types of frailty may be characterized by distinct metabolic perturbations. Amino acid profiling may therefore serve as a valuable tool for frailty biomarker discovery.