The objective of the study was to investigate the ability of an NMR-based metabonomic approach, applied to biofluids, to explore and identify overall exogenous and endogenous biochemical effects of a short-time high intake of milk protein or meat protein given to prepubertal children, the aim being to compare relative differences and not an absolute quantification. A total of twenty-four 8-year-old boys were asked to take 53 g protein as milk (n 12) or meat daily (n 12). At baseline and after 7 d, urine and serum samples were collected and high-resolution 1H NMR spectra were acquired on these using a 800 MHz spectrometer. The milk diet reduced the urinary excretion of hippurate, while the meat diet increased the urinary excretion of creatine, histidine and urea. The NMR measurements on serum revealed minor changes in the lipid profile, which most probably should be ascribed to an increase in the content of SCFA in the blood after consumption of the milk diet. The meat diet had no effect on the metabolic profile of serum. The study for the first time demonstrates the capability of proton NMR-based metabonomics to identify the overall biochemical effects of consumption of different animal proteins. The urine metabolite profile is more susceptible to perturbations as a result of short diet interventions than the serum metabolite profile. The milk diet-induced reduction in urinary excretion of hippurate suggests alterations in gut microflora, which may be useful information for further studies elucidating the effects of bioactive components in milk.