BACKGROUND: Alterations in glycolysis and oxidative pathways are central to the increasing incidence of non-alcoholic fatty liver disease (NAFLD), independent of body composition changes, highlighting a need for in vivo, non-invasive technologies to understand the development of hepatic metabolic aberrations in lean NAFLD. PURPOSE/HYPOTHESIS: To use hyperpolarized magnetic resonance spectroscopy (MRS) and proton density fat fraction (PDFF) MRI techniques to investigate effects of chronic, lifelong exposure to Western Diet (WD) in a model of NAFLD and identify cellular metabolite changes and correlations related to enzyme activity. It is hypothesized that exposure to the WD will result in NAFLD in association with altered pyruvate metabolism. STUDY TYPE: Prospective. POPULATION/SUBJECTS/PHANTOM/SPECIMEN/ANIMAL MODEL: 28 male guinea pigs were weaned onto a control diet or WD. FIELD STRENGTH/SEQUENCE: 3T; T1, T2, IDEAL, broadband PRESS MRS. ASSESSMENT: Median PDFF was calculated in the liver and hind limbs. [1-13C]pyruvate dynamic MRS in the liver was quantified by the time to peak (TTP), calculated as the time from pyruvate peak to metabolite peak. After a recovery period, animals were euthanized, and tissue was analyzed for lipid and cholesterol concentration and enzyme level and activity. STATISTICAL TESTS: Unpaired Student's t-tests were used to determine differences in measurements between the two diet groups. The Pearson correlation coefficient was calculated to determine correlations between measurements. RESULTS: Lifelong WD consumption resulted in significantly higher liver PDFF correlated with elevated triglyceride content in the liver. The WD group exhibited a decreased TTP for lactate production, and ex vivo studies highlighted increased liver lactate dehydrogenase (LDH) activity. DATA CONCLUSION: PDFF MRI results suggest differential fat deposition patterns occurring in animals fed a lifelong WD, corresponding with increased liver triglyceride levels characteristic of lean NAFLD. The decreased liver lactate TTP and increased ex vivo LDH activity suggest lipid accumulation occurs in association with a shift from oxidative metabolism to anaerobic glycolytic metabolism in WD livers.