This study presents an NMR-based metabonomic approach to explore the overall endogenous biochemical effects of a rye versus wheat-based fiber-rich diet in hypercholesterolemic pigs. The pigs were fed high-fat, high-cholesterol rye- (n = 9) or wheat- (n = 8) based buns with similar levels of dietary fiber for 9-10 wk. Fasting plasma samples were collected 2 days before and after 8 and 12 days on the experimental diets, while postprandial samples taken after 58-67 days, and( 1)H NMR spectra were acquired on these. Principal component analysis on the obtained NMR spectra demonstrated clear effects of diet on the plasma metabolite profile, and partial least squares regression discriminant analysis on the spectra revealed that the intensity of the spectral region at 3.29 ppm dominated the differentiation between the two diets, as the rye diet was associated with higher spectral intensity in this region. The 3.29 ppm signal is ascribed to N(CH(3))(3) protons in betaine, which may be an important contributor to the health promoting effects of rye.