OBJECTIVE Non-alcoholic fatty liver disease (NAFLD) is related to the metabolic syndrome and obesity. Proton magnetic resonance spectroscopy ((1)H MRS) is a non-invasive technique to assess hepatic triglyceride content (HTGC) and allows assessment of unsaturated fatty acids (UFA). There is increasing evidence that hepatic UFA are associated with the development of NAFLD. Therefore the objective of this study was to assess hepatic UFA in patients with NAFLD using (1)H MRS. METHODS We included 26 consecutive patients with deranged liver enzymes, with and without type 2 diabetes mellitus (DM2), suspected for NAFLD. Liver function and metabolic parameters were assessed. (1)H MRS measurements were performed at 3.0T. From the (1)H MR spectra two ratios were calculated: ratio 1 (UFA); unsaturated fatty acid peak vs. reference water peak and ratio 2 (HTGC); total fatty acid peak vs. reference water peak. RESULTS Twenty-six patients were included. In these patients hepatic UFA (ratio 1) correlated with AST/ALT ratio (r=-0.46, p=0.02), glucose levels (r=0.46, p=0.018), HOMA-IR (r=0.59, p=0.004) and HTGC (r=0.81, p<0.001). In diabetic patients (n=12) hepatic UFA correlated with alkaline phosphatase levels (r=0.72, p=0.01), HOMA-IR (r=0.73, p=0.01) and HTGC (r=0.83, p=0.002). Compared to non-diabetic patients with NAFLD, hepatic UFA levels were increased in patients with DM2 and NAFLD (0.032 vs. 0.014, p=0.03). CONCLUSIONS Hepatic UFA can be assessed with (1)H MRS. (1)H MRS determined hepatic UFA correlate with clinical and metabolic parameters associated with NAFLD. Hepatic UFA are increased in patients with DM2. This study provides evidence for the use of non-invasive (1)H MRS to assess hepatic UFA in vivo.