Consumers are increasingly concerned about the welfare of farm animals, especially during transportation. The Canadian transport regulations state that weaned cattle require an 8 h rest after 36 h of transport. There are, however, only a few studies that assess the effect of rest on the welfare of beef cattle. The aim of this study was to assess the effect of conditioning, source and rest on indicators of welfare in 7-8-month-old beef calves during long-distance transport. Treatments consisted of a 2 × 2 × 2 factorial design where the main factors were conditioning: conditioned (C; n = 160) or non-conditioned (N; n = 160); source: auction market (A; n = 160) or ranch direct (R; n = 160); and rest: 0 h (0 h; n = 160) or 8 h (8 h; n = 160). Means of non-esterified fatty acids (NEFA), serum amyloid-A, haptoglobin, creatine kinase (CK), and percentage of time standing from N calves were greater than C calves (all p ≤ 0.05). Means of percentage of time standing and CK of R calves were greater than A calves (both p ≤ 0.05). The mean of NEFA of 0 h calves was greater than the 8 h calves (p < 0.01), while the percentage of time standing of 0 h calves was less than 8 h calves (p < 0.01). Statistically significant differences between means of NEFA and standing percentage, were observed between 0 h and 8h calves. Few and inconsistent indicators of reduced welfare were observed between auction market and ranch direct calves, while non-conditioning was associated with greater physiological and behavioural indicators of reduced welfare. Based on these results, conditioning should be implemented as a management practice to improve the welfare of transported calves.