To obtain a better understanding of the progression of atherosclerosis and identify potential biomarkers, proton nuclear magnetic resonance spectroscopy (¹H NMR)-based metabonomics was used to study the metabolic changes in the plasma of hamster fed with a high-fat/cholesterol diet.  Plasma samples were collected at different time points during the progression of atherosclerosis and individual proton NMR spectra were visually and statistically assessed using multivariate analyses.  NMR results for all samples showed a time-dependent development from physiological to pathophysiological status during atherosclerosis.  Analysis of the identified biomarkers of atherosclerosis suggests that lipid and amino acid metabolisms are significantly disturbed, together with inflammation, oxidative stress, following cholesterol overloading.  The results enriched our understanding of the mechanism of atherosclerosis and demonstrated the effectiveness of the NMR-based metabonomics approach to study such a complex disease.