Recommendations for transportation of lambs, horses, calves, and pigs from a committee of the European Commission, which required rest stops of 6 or 24 h, every 8 h, were evaluated using Rambouillet x Suffolk lambs. The lambs of 17.6 +/- 0.5 kg of BW were randomly assigned to 1 of 3 treatments: transported for 22 h (continuous; n = 15); transported for 8 h, unloaded and rested for 6 h, transported for 8 h, unloaded and rested for 24 h, transported for 6 h (rested, n = 15); or remained in the home pasture throughout the study (control, n = 16). Off-trailer rest with food and water occurred in novel pens. Food deprivation in the continuous lambs was reflected by a decrease (P < 0.001) in plasma concentrations of glucose and an increase (P < 0.02) in plasma concentrations of blood urea nitrogen, creatinine, and total bilirubin relative to rested or control lambs. Electrolytes varied within and among all 3 treatments (P < 0.05), but no distinct pattern indicating dehydration was evident. Serum concentrations of cortisol were elevated in continuous and rested lambs compared with control lambs at 22 h (P < 0.05). Plasma immunoglobulin G antibody response to ovalbumin was suppressed (P < 0.05) in the continuous and rested lambs relative to the control lambs. Differences (P < 0.05) between continuous and rested lambs indicated the rest stops were sufficient to maintain BW during transport; however, these results were confounded by the control lambs losing a similar (P = 0.50) percentage of their initial BW as the continuous lambs at 22 h. The rest stops eliminated the physiological indicators of food deprivation and maintained BW but did not alleviate evidence of immunosuppression, and 52 h was required to complete the otherwise 22-h-long trip.