Background: The relationship between amino acids, B vitamins, and their metabolites with D3-creatine (D3Cr) dilution muscle mass, a more direct measure of skeletal muscle mass, has not been investigated. We aimed to assess associations of plasma metabolites with D3Cr muscle mass, as well as muscle strength and physical performance in older men from the Osteoporotic Fractures in Men cohort study.Methods: Out of 1 425 men (84.2 +/- 4.1 years), men with the lowest D3Cr muscle mass (n = 100), slowest walking speed (n = 100), lowest grip strength (n = 100), and a random sample (n = 200) serving as a comparison group to the low groups were included. Metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Metabolite differences between the low groups and random sample and their relationships with the muscle outcomes adjusted for confounders and multiple comparisons were assessed using t-test/Mann-Whitney-Wilcoxon and partial correlations, respectively.Results: For D3Cr muscle mass, significant biomarkers (p < .001) with >= 10% fold difference and largest partial correlations were tryptophan (Trp; r = 0.31), kynurenine (Kyn)/Trp; r = -0.27), nicotinamide (Nam)/quinolinic acid (Quin; r = 0.21), and alpha-hydroxy-5-methyl-tetrahydrofolate (hm-THF; r = -0.25). For walking speed, hm-THF, Nam/Quin, and Quin had the largest significance and fold difference, whereas valine (r = 0.17), Trp (r = 0.17), HKyn/Xant (r = -0.20), neopterin (r = -0.17), 5-methyl-THF (r = -0.20), methylated folate (r = -0.21), and thiamine (r = -0.18) had the strongest correlations. Only hm-THF was correlated with grip strength (r = -0.21) and differed between the low group and the random sample.Conclusions: Future interventions focusing on how the Trp metabolic pathway or hm-THF influences D3Cr muscle mass and physical performance declines in older adults are warranted.