Previous studies have shown that obese and overweight models have increased oxidative stress and lipid metabolism dysregulation. Thus, the objective of this study was to identify metabolite changes and potential biomarkers during weight loss that could be used as indicators of reestablished health. During a 4-wk baseline period, 8 adult neutered male cats were fed to maintain BW. For 18 wk following baseline, cats were fed to lose weight at a targeted rate of 1.5% BW/wk. As expected, mean BW (7.7 ± 0.4 vs. 6.2 ± 0.4 kg) and mean BCS (7.6 vs. 6.0) decreased (P < 0.05) at wk 18 vs. wk 0. Daily food intake, twice-weekly BW, and weekly BCS were recorded throughout the study. Fasted (~15 h) blood samples were collected at wk 0, 1, 2, 4, 8, 12, and 16. Serum was stored at −80°C until GC-MS and LC-MS/MS analyses. A total of 535 named biochemicals were identified, with up to 269 metabolites altered (P- and q- values <0.05) at wk 16 vs. wk 0. Principal component analysis showed a continual shift as weekly weight loss progressed. Component 1 and 2 explained 14.3% and 10.3% of the variability, respectively. There was a significant and dramatic reduction of bile acids (cholate; taurocholate; deoxycholate) with weight loss. A reduction in numerous free fatty acids (FFA) and an increase in ketones (acetoacetate; 3-hydroxybutryate) and monoglycerides suggest a shift toward lipolysis and hepatic FFA oxidation. Decreased markers of inflammation and oxidative stress were indicated by reduced pro-inflammatory oxylipids, eicosanoids, and oxidized biomarkers following weight loss. Markers of collagen degradation, such as proline-hydroxyproline and proline-glycine, and muscle protein turnover, such as 1-methylhistidine, increased. Mevalonate was significantly decreased after wk 8 compared with baseline, which agrees with the reduced bile acids without altering cholesterol. This may suggest upregulation of hepatic ketogenesis through activation of HMG-CoA lyase. Considering these results, global metabolomics was useful to potentially identify biomarkers of systemic changes in response to caloric restriction of a high-protein, high-fiber diet.