Background <p>Captive cheetahs are prone to a range of unusual diseases potentially linked to unnatural diets high in muscle meat and low in collagen-rich animal fibre. In the wild, cheetahs typically eat whole prey diets not easily replicated in a captive setting. Glycine is the most abundant amino acid in collagen with a key role in several metabolic pathways such as collagen biosynthesis. Several recent studies suggest that endogenous glycine production may be limited in several species.</p> Objectives <p>Using untargeted ¹H- nuclear magnetic resonance, the metabolic changes in the urine and serum of 10 adult captive cheetahs on a glycine-supplemented diet were investigated.</p> Methods <p>Cheetahs were fed either a meat only (control) or glycine-supplemented meat diet (30&#xa0;g glycine per 1&#xa0;kg meat) for four weeks, followed by a four-week cross-over. Urine and blood samples were collected at baseline and after each intervention.</p> Results <p>A total of 151 and 60 metabolites were identified in the urine and serum, respectively. Specifically, dimethylsulphone, proline, fructose, dimethylamine, trimethylamine, pyroglutamic acid, 1,3-diaminopropane, dihydrothymine, methylmalonic acid and pimelic acid contributed to metabolome differences in the urine. In serum, glutamic acid, threonine, α-aminobutyric acid, glucose-6-phosphate, ethanolamine, methionine and propionic acid were highlighted. These metabolites play various metabolic roles in energy production, immune function, protein and collagen biosynthesis or as products of gut microbiome fermentation.</p> Conclusion <p>Glycine supplementation influenced threonine sparing, pyrimidine biosynthesis pathways and bacterial fermentation products, although the implications of these findings on the health of captive cheetahs is unknown. Future studies should use a targeted approach to further elaborate on these pathways.</p>