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(1) Background: Ectopic fat deposition and its effects, metabolic syndrome, have been significantly correlated to lifestyle and caloric consumption. There is no specific noninvasive evaluation tool being used in order to establish clinical markers for tracing the metabolic pathway implicated in obesity-related abnormalities that occur in the body as a result of a high-fat diet (HFD). The purpose of this work is to investigate in vivo ectopic fat distribution and in vitro metabolite profiles given by HFDs, as well as how they are inter-related, in order to find surrogate metabolic biomarkers in the development of metabolic syndrome utilizing noninvasive approaches. (2) Methods: Male Wistar rats were divided into a standard normal chow diet, ND group, and HFD group. After 16 weeks of different diet administration, blood samples were collected for proton nuclear magnetic resonance (1H NMR) and biochemical analysis. Magnetic resonance imaging/proton magnetic resonance spectroscopy (MRI/1H MRS) was performed on the abdomen, liver, and psoas muscle of the rats. (3) Results: Visceral fat showed the strongest relationship with blood cholesterol. Although liver fat content (LFC) was not associated with any biophysical profiles, it had the highest correlation with metabolites such as (-CH2)n very-low-density lipoprotein/low-density lipoprotein (VLDL/LDL), lactate, and N-acetyl glycoprotein of serum 1H NMR. HFD showed no obvious influence on muscle fat accumulation. Acetoacetate, N-acetyl glycoprotein, lactate, (-CH2)n VLDL/LDL, and valine were the five possible metabolic biomarkers used to differentiate HFD from ND in the present study. (4) Conclusions: Our study has validated the influence of long-term HFD-induced ectopic fat on body metabolism as well as the metabolic profile deterioration both in vivo and in vitro. [ABSTRACT FROM AUTHOR]