The objective was to investigate the effect of 18h road transport with a 12h mid-journey rest period in comparison to the exposure of bulls to a novel environment on physiological, metabolic and behavioural responses of beef bulls. Thirty Charolais sired crossbred beef bulls (mean 486.0, s.d. 57.0kg) were assigned by live weight to one of the two treatments, transport (T) (9h+9h) (n=15) and not-transported (NT) (n=15) on day 0. The bulls were transported at a spatial allowance of 1.3m(2)/animal by road for 9h, unloaded and rested for a 12h rest period, re-loaded and transported for a further 9h journey by road followed by a 2h rest period on the transporter, then unloaded and rested in a lairage for 24h with access to hay and water. Plasma albumin and urea concentrations increased (P<0.05) after the first 9h journey with values returning to baseline at the end of the 24h recovery period. There was a transient increase in haematocrit% in T and NT at sampling time points corresponding to the completion of the first 9h journey. Bulls spent longer time lying (P<0.05) during the first 9h journey compared with the percentage time spent lying during the second 9h journey. Differences in live weight, behaviour, and some blood variables show that transport is more stressful for bulls than being subjected to a novel environment and management, and while some biological variables returned to baseline values, others require a longer time (plasma haptoglobin, total protein, glucose and NEFA concentrations). Thus, the effective recovery of bulls exposed to an 18h transport journey by road would suggest that a rest period of at least 24h with access to feed and water is required before further transport.