Glucagon-like peptide 1 receptor (GLP-1R) agonists and dual agonists targeting GLP-1R and the glucagon receptor (GCGR) or the glucose-dependent insulinotropic peptide receptor (GIPR) are currently being developed for the treatment of non-alcoholic steatohepatitis (NASH). We have tested specific mono-agonists to these three receptors individually and in combination in a mouse model of diet-induced NASH and fibrosis, to decipher the contribution of their activities and potential additive effects on improving systemic and hepatic metabolism.NASH was induced by pre-feeding C57BL/6J mice a diet rich in fat, fructose and cholesterol for 36 weeks. This was followed by eight weeks of treatment with the receptor-specific agonists 1-GCG (20 μg/kg b.i.d.), 2-GLP1 (3 μg/kg b.i.d.) or 3-GIP (30 μg/kg b.i.d.), or the dual (1 + 2) or triple (1 + 2 + 3) combinations thereof. A dual GLP-1R/GCGR agonistic peptide, 4-dual-GLP1/GCGR (30 μg/kg b.i.d.), and liraglutide (100 μg/kg b.i.d.) were included as references.Whereas low-dose 1-GCG or 3-GIP alone did not influence body weight, liver lipids and histology, their combination with 2-GLP1 provided additional weight loss, reduction in liver triglycerides and improvement in histological disease activity score. Notably, compared to high-dose liraglutide, 4-dual-GLP-1R/GCG and the triple combination of selective mono-agonists demonstrated stronger reduction in NAFLD activity score at the same extent of weight loss.GCGR and GIPR agonism provide additional, body weight-independent improvements on top of GLP-1R agonism in a murine model of manifest NASH with fibrosis. This article is protected by copyright. All rights reserved.