PURPOSE: Adipocyte cell size varies among individuals and importantly, is inversely correlated with insulin sensitivity, and modifiable by weight loss or pharmaceutical agents. However, there are no non-invasive, in vivo methods for adipocyte cell size determination. Here we apply Chemical-Shift Water-Fat MRI to in vivo measures of subcutaneous (inguinal) and visceral (gonadal) white adipose tissue (WAT) to determine whether the fat-signal fraction (FF) is a sensitive indicator of adipocyte cell size. MATERIALS AND METHODS: C57BL/6J male mice (8 weeks old) were singly housed and fed a low-fat diet, high-fat diet or very high-fat diet (n = 16 or 15/group) for 8 weeks. Food intake, body weight and composition were measured; CS-MRI was performed on a 9.4 Tesla Bruker magnet with respiratory gating and anesthesia. Histology was acquired for gonadal WAT; both gonadal and inguinal WAT were fixed with osmium tetroxide and then measured through Image J for cell size. RESULTS: Mice fed with higher fat content diets gained significantly more body weight, fat and lean mass while maintaining higher energy intakes over the 8 weeks. There was no significant difference in fat fraction for either gonadal (P = 0.1295) or inguinal (P = 0.4704) WAT among the three groups, despite significantly larger adipocytes (P <0.0001) in mice on high fat diets. CONCLUSION: Although diet-induced obesity significantly increased the amount of fat mass, as well as mean and overall white adipocyte cell size, the CS-MRI measured fat fraction between groups were not significantly different. These results do not support the utility of CS-MRI measured FF for in vivo determination of adipocyte cell size.