Introduction <p>Analysis of specific pathways of metabolic flux is usually achieved by administering stable isotope-labeled precursors and measuring their incorporation into selected metabolites by high-resolution mass spectrometry coupled to liquid chromatography (LC-HRMS).</p> Objectives <p>In this study, we undertook a novel approach aiming at covering the whole metabolome dynamics by providing mice with a diet fully enriched in carbon-13 (¹³C).</p> Methods <p>Three animals were fed for six weeks with small pellets composed of a mixture of ¹³C-spirulina and ¹³C-wheat. All animals grew normally and their urine was collected daily. Three control mice were treated in the same way, but with an unenriched diet. LC-HRMS-based metabolomics profiling were conducted on all collected samples as well as isotope tracing.</p> Results <p>Comparative LC-HRMS analysis of the ¹²C- and ¹³C-samples unambiguously identified 238 metabolites, whose ¹³C-labeling profiles were then studied over a 39-day period. Although overall urine labeling was fast and rapidly reached a high level of ¹³C-content (&gt; 90% after 22 days), isotopic monitoring of each molecular species demonstrated that ¹³C-incorporation kinetics are considerably variable between metabolites, reflecting their biological function, origin and rate of biosynthesis in vivo.</p> Conclusion <p>This study demonstrates that mice tolerate a diet entirely labeled with carbon-13 (&gt; 97 atom % ¹³C) over a period of six-weeks. It reveals the dynamics of a large part of the metabolome at the mammalian species level through urine analysis coupled to in vivo ¹³C-labeling. The study provides valuable insights by comprehensively covering metabolic pathways, and stands out from targeted fluxomic studies which are carried out by administering a very limited number of specific labeled precursors.</p>