Twenty unweaned Angus-Holstein calves (44.1 ± 2.0 kg; 14.7 ± 0.63 d) were used to evaluate the effects of feed restriction and fasting on energy balance (serum concentration of glucose, BHBA, and NEFA), and intestinal permeability (serum concentration of citrulline, Cr-EDTA, lactulose, and D-mannitol). Calves were randomly assigned to 1 of 4 treatments that simulated assembly center management and transportation; Control (CT; n = 5): from d-4 to -1, 2.5 L of milk replacer (MR) twice daily, concentrate and straw ad-libitum; Mild (MD; n = 5): only MR (d-4 to -1) as described for CT, and on d-1 feed withdrawal for 9 h; Moderate (MO; n = 5): only MR as described for CT and on d-1 feed withdrawal for 19 h; and Severe (SV; n = 5): only 2.5 L of a hydrate solution (HS) twice daily (d -4 to -1) on d-1 feed withdrawal for 19 h. From d 0 to day 7 all calves were fed MR, concentrate, and straw ad-libitum. Data were analyzed using mixed models with repeated measures. At d -1 for SV and d 0 in all restricted calves (MD, MO, SV), serum glucose concentration was lesser (P < 0.01) compared with CT. At d 0 NEFA and BHBA serum concentrations increased (P < 0.01) in SV calves compared with the other treatments. Serum citrulline concentration (P < 0.05) was lower for SV on d -1 and greater for CT on d 2 compared with other treatments. CT calves had the lesser concentration of Cr-EDTA (d -1 and d 0, P =0.04), lactulose (d 0, P =0.02), and D-mannitol (d 0, P =0.01) compared with MD, MO, and SV. The degree of dietary restriction, the type of liquid diet, and amount of fasting hours affected calves’ serum concentration of markers indicative of energy balance and gut permeability.