The rising prevalence of childhood obesity imposes a heavy socioeconomic burden globally. Weight loss strategies have varied in their effectiveness. Understanding metabolism is crucial in designing weight loss diets for children and adolescents to ensure they achieve their goals safely, without compromising their growth, development, or overall health. This study aimed to explore the metabolic impact of weight loss in children with obesity (BMI of 20.71 to 43.84) following a 2-5 week healthy lifestyle programme undertaken at a residential camp in the UK. Spot urine samples (n = 103) were collected from 36 children (age 8-15) at baseline and on a weekly basis throughout the intervention duration and analysed using 1H Nuclear magnetic resonance (NMR) spectroscopy. Spectra were modelled in relation to % weight change, % BMI change and weekly change in metabolic profile using an orthogonal projection to latent structure (OPLS) method. Clear metabolic differences post intervention were identified at 1 week post intervention with increased excretion of gut microbial metabolites (hippurate, 4-cresylsufate, phenylacetylglutamine), N-methylnicotinamide, ketone bodies, and the previously unreported metabolite trans-3-hexeneoyl glutamine. The structure of this metabolite was elucidated using a combination of statistical correlation spectroscopy (STOCSY), along with one and two dimensional NMR analyses, LC-MS/MS analyses, total synthesis, and matrix spiking. A detailed account of the structural elucidation process is presented herein.