The 1H NMR spectra of low-volume plasma samples could be acquired by NMR spectrometer and using 3 mm NMR sample tubes. This strategy is essential for the limited availability of samples, such as plasma samples from mice or children. Thus, we investigated the effects of a high-fat diet on Apo-E-/- mice via the utility of low sample volume in proton nuclear magnetic resonance (-H NMR) experiments. The 1H1 NMR spectra, including 1D solvent-presaturation, and CPMG spin−echo, were acquired and applied for 33 metabolites and 112 lipoproteins subclass analysis. In lipoprotein subclass analysis, we acquired data that includes cholesterol, free-form cholesterol, triacylglycerol, and phospholipid in individual lipoprotein subfractions (VLDL, IDL, LDL, and HDL) of plasma samples. The total triacylglycerol and cholesterol values in Apo-E-/- mice were higher than in wild-type mice. However, there is only cholesterol and phospholipid enhanced in high-fat diet-treated mice. Furthermore, the lipoprotein profiles revealed that the high-fat diet enhanced cholesterol, free-form cholesterol, and phospholipids accumulation in LDL subclass lipoprotein of Apo-E-/- mice but in HDL of wild-type mice. The Apo-B in the LDL subfraction (LDL-1, LDL-2, and LDL-3), and Apo-A in the HDL subfraction of HDL-4 were enhanced in high-fat diet-treated Apo-E-/- mice. The 3 mm NMR tube and lipoprotein subclass analysis were suitable for volume-limited samples of lipoprotein subclass quantitation and low molecular weight metabolites profiling, which could be applied in mice disease model research.