The response to a one-hour road journey was observed in 145 semi-feral ponies unused to handling. Ponies were transported singly or in groups of four or eight at a typical commercial ‘high’ stocking density or at a slightly reduced ‘low’ density (range 139 to 316 kg m−2). Blood samples were obtained for plasma biochemistry post transport. Behaviour during the journey of 40.2 miles was recorded using overhead video cameras. Analysis of these revealed that levels of aggression were lower in groups of four, as were the number of slips, falls, stumbles and collisions (SFSC). Within groups of four or eight, reduced stocking density was associated with lower levels of plasma cortisol and creatine kinase, reduced aggression and fewer SFSC. Levels of aggression pre-transit (observed over one hour the day before travel) were not predictive of aggression on the lorry. Single ponies were constrained to align at right angles to the direction of transport, whereas ponies transported in groups could also align forwards or backwards and were able to re-align themselves during transport especially at reduced stocking density. Of those transported singly, younger ponies showed separation anxiety, with highly-significantly greater levels of pawing and sniffing at the ground and increased packed cell volume. Thus, transporting young, untamed ponies singly is not recommended. A suitable maximum stocking density for ponies transported in groups would be approximately 200 kg m−2. However, minimum stocking density for grouped and single untamed ponies transported by road requires future investigation. Whilst stocking density was correlated with many of the variables measured during transport, stocking rate (animals per unit floor area) was not.