Hepatic steatosis was rapidly induced in both NASH- and HFD-fed rats and was shown using qCT to progress in a time-dependent manner. In line with this, higher levels of plasma β-hydroxybutyrate were observed in HFD-fed rats, suggestive of increased β-oxidation due to hepatic lipid overload. The role of hepatic β-oxidation in the pathogenesis of NAFLD remains controversial, but several studies in human NASH-patients have found serum levels of β-hydroxybutyrate to be increased [24, 25]. Although not significant, there was a trend towards an increase of this marker in NASH-fed rats as well. The development of hepatic steatosis in NASH- and HFD-fed rats agrees well with recent studies in other rodent species suggesting fat and cholesterol to be important drivers in the development of experimental NAFLD/NASH [14, 22]. The addition of dietary cholesterol (NASH-diet) caused the most detrimental liver changes, evident by significant accumulation of both hepatic TG and cholesterol, pronounced decreases in liver density, and severe morphological alterations in liver histology. In contrast to what was observed in HFD- and HFr-fed rats, these changes were also accompanied by increased plasma levels of ALAT and ASAT, suggesting impaired liver function. It has previously been observed in both mice [26, 27] and humans  that the combined intake of dietary fat and carbohydrates, or of fat and cholesterol  can result in accelerated and more severe effects in the liver. The majority of these changes could be driven by the amount of cholesterol in the NASH-diet (2%), as hepatic levels of cholesterol measured in NASH-fed rats were very high. Supporting this, two previous studies in mice have suggested that dietary cholesterol is important in facilitating progression from simple steatosis to NASH  and that free cholesterol loading can sensitize the liver to cytokine-mediated hepatocellular death, inflammation and oxidative stress . This could account for the more aggravated inflammatory histological response, supported by the significant increase in infiltrating macrophages observed in livers of NASH-fed rats compared to both HFD and Control groups. Further studies are needed to determine if these changes are caused exclusively by the cholesterol content, by a synergistic effect of fat and fructose, or by a combination of all.