This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLSDA) on spectra obtained for plasma samples revealed that the spectral region at 3·25 parts per million dominates the differentiation between the two diets, as the WGD is associated with higher spectral intensity in this region. Spiking experiments and LC–MS analyses of the plasma verified that this spectral difference could be ascribed to a significantly higher content of betaine in WGD plasma samples compared with NWD samples. In an identical study with the same diets, urine samples were collected, and1H NMR spectra were acquired on these. PLS-DA on spectra obtained for urine samples revealed changes in the intensities of spectral regions, which could be ascribed to differences in the content of betaine and creatine/creatinine between the two diets, and LC–MS analyses verified a significantly lower content of creatinine in WGD urine samples compared with NWD urine samples. In conclusion, using an explorative approach, the present studies disclosed biochemical effects of a wholegrain diet on plasma betaine content and excretion of betaine and creatinine.