To evaluate the effect of journey duration and space allowance on behavioural and physiological responses, 72 male suckling lambs were transported by road to the slaughterhouse using two transport times - short (around 40min) or long (around 4.75h) - and three different space allowances 0.12 (LSA), 0.20 (MSA) and 0.25 (HSA)m 2/lamb. Lamb behaviour was recorded continuously during the entire journey. A heart rate monitor was used to determine the heart rate during transport, and blood samples were collected two days prior to transport and after unloading at the slaughterhouse to measure the concentrations of various stress-related variables. Immediately after slaughter, and 24h thereafter, samples of Musculus longissimus were collected to determine muscle glycogen concentrations and inosine monophosphate (IMP)/adenosine triphosphate (ATP) ratio. The highest number of lambs was standing for both short and long journeys, whereas the lowest number was walking. However, the number of lambs performing each behaviour was found to differ with journey time, with significant differences in the behaviour shown by lambs transported at different space allowances. The heart rate increased during loading with respect to the values recorded during the resting period on the farm for both journey times in lambs transported at LSA and MSA, and remained high throughout the transportation period for these two space allowances. Cortisol, LDH, glucose, albumin and osmolality were higher in suckling lambs transported for a short time than for those transported for longer periods. Space allowance did not affect any of the blood parameters studied except for albumin and plasma total protein concentration, both of which were lower for MSA group (0.20m 2/lamb). In addition, liver glycogen concentration was higher for lambs after the long journey. Our results suggest that short journey produce higher stress response in suckling lambs than long journey, due to mainly to loading and initial transport which were similar handling and transport to first part of long journey. Nevertheless, this initial stress response decreases during the journey, although the heart rate remains high throughout mainly as space allowance decrease. © 2012 Elsevier B.V.