The aim of this study was to assess the effects of conditioning, rest, and post-rest transport duration on welfare indicators of 6–7 mo old beef calves following a 20-h transport. Three hundred and twenty-eight weaned calves (237 ± 29.7 kg of BW) were randomly assigned to a 2 × 2 × 2 nested factorial design: conditioning, conditioned (C) or non-conditioned (N); rest, 0 (R0) or 8 (R8) h, and post-rest transport, 4 (T4) or 15 (T15) h. Calves were sampled before (LO1) and after (UN1) the initial 20-h journey, before (LO2) and after (UN2) the additional 4 or 15-h journey, and at 1, 2, 3, 5, 14, and 28 d after UN2. Data was analyzed using the GLIMMIX procedure of SAS. Fixed effects included conditioning, transport, and time nested within rest period, while random effects included animal and pen. Greater shrink (p < 0.01) was observed in C than N calves after the initial 20-h transport. During the first week after transportation, the mean ADG of N calves was greater than C calves (p < 0.01). From d 14 to d 28, however, the mean ADG of C calves was greater than N calves (p < 0.01). Flight speed, cortisol and L-lactate concentrations were greater (p ≤ 0.05) in C than N calves between LO1 and d 5, while greater (p ≤ 0.02) non-esterified fatty acids, creatine kinase, serum amyloid-A, and haptoglobin concentrations were observed in N than C calves between LO1 and d 3. The R8-T4 calves had greater (p < 0.01) ADG than R8-T15 calves between LO1 and d 5. The R0-T4 calves had greater L-lactate concentrations than R0-T15 and R8-T4 calves (both p = 0.02) on d 1. The R0 calves had greater (p < 0.01) ADG than R8 calves between 14 and 28 d. This study suggests that C calves are better fit for transport than N calves as evidenced by behavioural and physiological parameters. Fewer and inconsistent differences were observed for rest and post-rest transport treatments.info:eu-repo/semantics/publishedVersio