Background Currently it is unclear whether lipid accumulation occurs in a particular sequence and its relationship with whole body insulin resistance (IR). This study aimed to answer this question. Methods Male Sprague-Dawley (SD) rats were fed on a normal or a high-fat diet for 20 weeks. Serum triglycerides (TG), serum free fatty acids (FFA), fasting plasma glucose (FPG), and liver and skeletal muscle TG were measured. The glucose infusion rate (GIR) and mRNA levels of acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase-1 (CPT-1) in the liver and skeletal muscle were determined at different stages. Results Compared with rats fed on the normal diet, serum FFA was not significantly increased in rats fed on the high-fat diet until 20 weeks. In contrast, liver TG was significantly increased by the high-fat diet by four weeks (20-fold; P<0.01), and remained elevated until the end of the study. However, skeletal muscle TG was not significantly increased by the high-fat diet until 20 weeks (10.6-fold; P<0.01), and neither was the FPG. The GIR was significantly reduced (1.6-fold; P<0.01) by the high-fat diet after 8 weeks. The mRNA levels of ACC gradually increased over time and CPT-1 decreased over time, in both the liver and skeletal muscle in rats fed the high-fat diet. Conclusions Lipid accumulation in the liver occurs earlier than lipid accumulation in the skeletal muscle. Fatty liver may be one of the early markers of whole body IR. Changes in the gene expression levels of ACC and CPT-1 may have important roles in the process of IR development. Chin Med J 2010;123(24):3605-3611