Purpose To prospectively evaluate the changes in fatty acid concentration after administrating a 60% high-fat diet to a non-alcoholic fatty liver disease rat model and to perform a correlation analysis between fatty acid with molecular diffusion (D-true), perfusion-related diffusion (D-fast), and perfusion fraction (P-fraction). Material and Methods This prospective study was approved by the appropriate ethics committee. Ten male Sprague-Dawley rats were fed a 60% high-fat diet until the study was finished. Point-resolved spectroscopy sequence H-1-MRS with TR = 1,500 msec, TE = 35 msec, NEX = 64, and 8x8x8 mm(3) voxel was used to acquire magnetic resonance spectroscopy (MRS) data. Diffusion-weighted imaging was performed on a two-dimensional multi-b value spin echo planar image with the following parameters: repetition time msec/echo time msec, 4500 /63; field of view, 120x120 msec(2); matrix, 128x128; section thickness, 3 mm; number of repetition, 8; and multiple b value, 0, 25, 50, 75, 100, 200, 500, 1000 sec/mm(2). Baseline magnetic resonance imaging and magnetic resonance spectroscopy data (control) were acquired. H-1 proton MRS and diffusion-weighted imaging were obtained every 2 weeks for 8 weeks. The individual contributions of the true molecular diffusion and the incoherent motions of water molecules in the capillary network to the apparent diffusion changes were estimated using a least-square nonlinear fitting in MatLab. A Wilcoxon signed-rank test with the Kruskal-Wallis test was used to compare each week's fatty acid mean quantification. Spearman's correlation coefficient was used to evaluate the correlation between each fatty acid (e.g., total lipid (TL), total saturated fatty acid (TSFA), total unsaturated fatty acid (TUSFA), total unsaturated bond (TUSB), and polyunsaturated bond (PUSB)) and intravoxel incoherent motion (IVIM) mapping images (e.g., D-true, D-fast, and P-fraction). Results The highest mean TL value was at week 8 (0.278 +/- 0.10) after the administration of the 60% high- fat diet, followed by weeks 6, 4, 2, and 0. The concentration level (16.99 +/- 2.29) of TSFA at week 4 was the highest. No significant differences in the concentrations of TUSFA, TUSB, or PUSB were observed in different weeks. Conclusion After the administration of the 60% high-fat diet in nonalcoholic fatty liver disease model, TL and TSFA depositions had significant changes. The mean concentrations of TUSFA, TUSB, PUSB did not significantly change. Total unsaturated fatty acid and polyunsaturated bond showed positive correlations with D-true and P-fraction.