We previously found that a short-term restriction of 70% dietary Mg did not alter whole body insulin sensitivity but resulted in hepatic insulin resistance in growing rats fed a high-fat (HF) diet (Clin. Nutr 33(5): 879-88, 2014). Here, we evaluated time-dependent changes in whole body insulin sensitivity in HF rats submitted to a restriction of 90% dietary Mg for a longer time of intervention (16 weeks). Male Wistar rats (n = 24; initial body weight 97-123 g) received ad libitum semipurified and pelleted AIN-93-based control (CT) or HF diets (adequate or Mg-restricted; 500 and 50 mg Mg/kg diet, respectively). Body weight and food intake were recorded every three days. Insulin sensitivity was measured by an intraperitoneal insulin tolerance test at weeks 8, 12 and 16. The effects of Mg restriction on insulin sensitivity of HF-fed animals were significant after 16 weeks of experiment. At this time, food intake was decreased by HF (p < 0.01), but not by Mg restriction. As expected, Mg intake was about 87-90% lower in Mg-restricted rats comparatively to their respective control ones. Interestingly, body weight did not differ among groups. In contrast, Mg restriction resulted in insulin resistance in HF compared to control animals (CT[Mg500]) (p = 0.007), without affecting glucose homeostasis. Spearman´s correlations analysis revealed significant associations of insulin resistance (area under the 40-min blood glucose response curve [AUC]) with Mg intake (r = -0.47, p = 0.02) and body weight (r = 0.59, p = 0.002). In conclusion, our preliminary data indicate that the combination of two nutritional challenges (lipid overload and Mg restriction) for a longer period (16 weeks) led to whole-body insulin resistance in Wistar rats. The implications of these findings on the insulin resistance of target tissues related to Mg homeostasis are currently being investigated in our laboratory.