Metabolomics has been recognized as a powerful approach for disease screening. In order to highlight potential health issues in subjects, a key factor is the possibility to compare quantitatively the metabolome of their biofluids with reference values from healthy individuals. Such efforts towards the systematic characterization of the metabolome of biofluids in perfect health conditions, far from concluded for humans, have barely begun on horses. The present work attempts, for the first time, to give reference quantitative values for the molecules mostly represented in the urine metabolome of horses at rest and under light training, as observable by 1H-NMR. The metabolome of ten trotter horses, four male and six female, ranging from 3 to 8 years of age, has been observed by 1H-NMR spectroscopy before and after three training sessions. We could characterize and quantify 54 molecules in trotter horse urine, originated from diet, protein digestion, energy generation or gut-microbial co-metabolism. We were able to describe how gender, age and exercise affected their concentration, by means of a two steps protocol based on univariate and robust principal component analysis.