Numerous studies have indicated magnetic resonance (MR) imaging and proton MR spectroscopy (¹H MRS) as non-invasive tools to quantify lipids.¹ An increase in signal-to-noise (SNR) for metabolite quantification shortens the total acquisition time of lipid data and increases spectral resolution, thereby improving quantification of metabolites and relaxation times.² The measurement of relaxation time depends on fatty acid molecules and their degrees of unsaturation.³ Lipid content that is measured by ¹H MRS is thought to play an important pathogenic role in conjunction with changes in saturated and unsaturated fatty acids. These analytical methods are crucial for effective therapeutic interventions and strategies against NAFLD. The objective of this study was to determine the metabolic changes in a rat model of high-fat-diet-induced NAFLD by using single-voxel ¹H-MRS.The examinations were performed on a 3.0 T scanner (Achieva Tx 3.0 T; Philips Medical Systems, The Netherlands) using a 4-­channel animal coil for higher resolution. This coil was used for MR spectroscopy. This method used point-resolved spectroscopy (PRESS) (repetition time/echo time= 6000/35 ms; number of signal averages= 64). Shimming was performed by iterative VOI, and full width at half maximum (3 - 8 Hz) was achieved. Male Sprague-Dawley rats (n = 10) weighing 100-150 g were housed with ad libitum access to water and HF diet for 15 weeks. The HF diet pellets contained 60% fat, 20% protein, and 20% carbohydrate. In order to avoid large blood vessels, a voxel (0.8×0.8×0.8 cm³) was placed in a homogeneous area of the liver parenchyma during free breathing. To measure the lipid content, we quantified total lipids ((-CH2-)_n/noise), total saturated fatty acids (3(-CH₂-)/2(-CH₃)), total unsaturated fatty acids (3(-CH₂-C=C-CH₂-)/4(-CH₃)), total unsaturated bonds index (3(-CH=CH-)/2(-CH₃)), and polyunsaturated bonds index (3(=C-CH₂-C=) / 2(-CH₃)) by separating each peak area of (­CH₂­)_n, ­CH₂­-C=C-­CH₂­, =C-­CH_­2­-C=, and ­CH=CH­ by ­CH₃. The -CH₃ (0.90 ppm) peak was used as an internal chemical shift reference. Each peak was also scaled to the number of protons contributing to the resonance. Total lipid content was measured by dividing the (-CH_­2-)_n (1.30 ppm) peak area by spectral noise acquired with the standard deviation of peak area (from 7.30 to 11.30 ppm) when no lipid metabolites were detectable in the liver. All statistical analyses were conducted using the Statistical Package for the Social Sciences (SPSS version 21, IBM Corp. Armonk, NY, USA) using the independent t-test to determine metabolic changes and to identify significant differences relative to the baseline.Fig. 1 illustrates high spectral resolution and strong signals of methyl protons, methylene proton, allylic protons, diallylic protons, and methane protons. According to Fig. 2A and B, total lipids and total saturated fatty acids were substantially upregulated in high-fat-diet-fed rats in comparison with baseline values (total lipids, 16.22±6.60×10^-4; total saturated fatty acids, 10.26±1.91). The resulting data on lipid accumulation were statistically significant in terms of total lipids at 3 weeks (78.65±36.20), 6 weeks (88.14±22.36), 9 weeks (78.55±31.29), and 15 weeks (107.87±39.95) after initiation of the high-fat diet. There were early metabolic changes that appeared at 3 weeks. Compared to the baseline level, the total level of saturated fatty acids increased after 3, 6, 9, and 15 weeks of the high-fat diet. Early metabolic changes appeared at 3 weeks (14.77±3.97). According to Fig. 2C, compared to baseline values (0.39±0.18), the changes in total unsaturated fatty acids were statistically significant only at 9 weeks (0.63±0.23); the changes at 3 weeks, 6 weeks, and 15 weeks were not significant. According to Fig. 2D, compared to the baseline values (0.47±0.22), the change in the total unsaturated bonds index was statistically significant only at 15 weeks (0.59±0.35). At 9 and 15 weeks, this index decreased statistically significantly. According to Fig. 2E, compared to baseline values (0.52±0.26), the changes in the polyunsaturated bonds index were statistically significant between 3 weeks (0.99±0.39) and 9 weeks (0.83±0.24).In our study, although total unsaturated fatty acids in the liver show no significant changes until 6 weeks from the baseline values, a significant increase was observed after 9 weeks of high-fat diet. Similarly, a significant increase in the number of polyunsaturated bonds was observed after 3 and 9 weeks. Our study suggests that unsaturated fatty acids may be upregulated or downregulated in a chronic model of NAFLD. In conclusion, this study of ¹H MRS shows sufficient spectral resolution and SNR for the characterization of observable total lipids and fatty acids. Our results of noninvasive in vivo MRS are based on accurate monitoring of the changes in lipid content, which were verified using the data on saturated fatty acids and unsaturated fatty acids.