Abstract Introduction Bed-rest (BR) reduces whole-body insulin-stimulated glucose disposal (GD) and alters muscle fuel metabolism. However, little is known about metabolic adaptation from acute to chronic BR, particularly when volunteers are maintained in energy balance. Methods Healthy males (n=10, 24±1.25 years) maintained in energy balance underwent 3 days of BR (acute BR; ABR). A second cohort matched for gender and body mass index (n=20, 34±1.8 years) underwent 56 days of BR (chronic BR; CBR). A hyperinsulinaemic euglycaemic clamp (60 mU/kg lean mass/min) was performed before and after BR. Indirect calorimetry was performed before and during the clamp steady-state to calculate rates of whole-body fuel oxidation. Vastus Lateralis muscle biopsies were taken before and after each clamp. Two-way repeated measures ANOVA was used to detect differences in end-point measures. Results ABR reduced insulin-mediated glucose disposal (GD; p<0.001), which was unchanged in magnitude following CBR (p<0.05). This reduction in GD following both acute and CBR was paralleled by the elimination of a 35% increase in insulin-stimulated muscle glycogen storage seen Pre BR. ABR had no impact on insulin-stimulated carbohydrate (CHO) and lipid oxidation, but CBR reduced CHO oxidation (p<0.05) and blunted the magnitude of insulin-mediated inhibition of lipid oxidation (p<0.05). Neither acute nor CBR increased muscle intramyocellular lipid content. Conclusion ABR suppressed insulin-stimulated GD and glycogen storage, and the extent of suppression increased no further after CBR. However, GD and storage were dissociated from substrate oxidation during CBR. Moreover, the shift in substrate oxidation after CBR was not explained by IMCL accumulation. Take home message Acute bed rest impairs insulin-stimulated glucose disposal and glycogen storage which is the same magnitude as that seen in chronic bed rest.