Human MASLD is a diurnal disease driven by multisystem insulin resistance and reduced insulin availability at night.

Hepatic lipid and glucose metabolism have been shown to be under tight circadian control in pre-clinical models. However, it remains unknown whether diurnal patterns exist in functional processes governing intrahepatic lipid accumulation in humans. We performed metabolic phenotyping, including state-of-the-art stable isotope techniques, during day and night in patients with metabolic dysfunction-associated steatotic liver disease (MASLD) and overweight controls (NCT05962099). The primary outcome was diurnal change in hepatic de novo lipogenesis (DNL), alongside a number of secondary outcomes, including changes in hepatic glucose production, glucose disposal, plasma non-esterified fatty acids (NEFAs), and whole-body glucose and lipid oxidation. We show that nighttime metabolic dysfunction is a hallmark of MASLD with multiple pathogenic pathways upregulated at night, including hepatic and peripheral insulin resistance, DNL, and systemic NEFA exposure. Insulin resistance is compounded by lower plasma insulin levels at night, secondary to reduced insulin secretion and elevated insulin clearance. Diurnal differences persist when performing identical investigations after weight loss with liver fat reductions, suggesting that nighttime metabolic dysfunction may be a primary driver of steatosis. These findings will help establish the optimal window for energy intake, exercise, and medication delivery in patients with MASLD. Integrated proteomics of plasma, adipose, and skeletal muscle tissue across day and night also identified a number of specific molecular targets that may offer therapeutic potential in the treatment of metabolic disease.