While exercise is generally beneficial for the amelioration of Metabolic dysfunction-associated steatotic liver disease (MASLD), little is known about the best timing of exercise. Here we demonstrate in APOE*3-Leiden.CETP mice, an MASLD model, that specifically late, but not early, active period aerobic exercise leads to fat loss, amelioration of MASLD development and lower liver lipids. These changes coincide with a change in the gut microbiota composition of only late exercising mice towards a short-chain fatty acids producing profile. A fecal microbiota transplantation from late exercising mice to sedentary mice has partially reproduced MASLD-ameliorating effects of late exercise, depending on the success of the transplantation. These findings demonstrate the importance of the timing when conducting lifestyle interventions and lay a foundation for future clinical trials which can be used to adapt clinical guidelines for MASLD treatment.. Timing matters: Late, but not early, exercise

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) affects two billion people worldwide and is currently mostly treatable via lifestyle interventions, such as exercise. However, it is unclear whether the positive effects of exercise are restricted to unique circadian windows. We therefore aimed to study whether the timing of exercise training differentially modulates MASLD development. Methods: Twenty to twenty-six weeks old male APOE*3-Leiden.CETP mice were fed a high fat-high cholesterol diet to induce MASLD and trained on a treadmill for one hour five times per week for twelve weeks either early (ZT13; E-RUN) or late (ZT22; L-RUN) in the dark phase, while corresponding sedentary groups (E-SED and L-SED) did not. To assess the role of the gut microbiota in training-induced effects, the study was repeated and trained (ZT22 only, RUN) or sedentary mice (SED) served as fecal donors for sedentary recipient mice (RUN FMT and SED FMT). Results: In the first study, late but not early exercise training decreased the MASLD score, body weight, fat mass, and liver triglycerides, accompanied by altered composition of the gut microbiota. Specifically, late but not early exercise increased the abundance of short-chain fatty acid-producing bacteria families and genera, such as Akkermansia, Lachnospiraceae and Rikenella. Fecal microbiota transplantation reduced liver weight and plasma triglycerides in RUN FMT group compared to SED FMT, and tended to lower the MASLD score and liver triglycerides. Conclusion: Timing of exercise training is a critical factor for the positive effect on MASLD in this pre-clinical model, and the effect of late exercise is at least partially mediated via the gut-liver axis.