Long-distance transport can cause stress to beef cattle impairing health and growth performances. This study aimed to deepen the knowledge of the effects of long-distance transport on young bulls’ metabolic profile to identify reliable blood parameters for monitoring their health and welfare. Eighty Charolais young bulls, transported for 12 hours to the final fattening unit, were weighed and blood sampled at three time intervals: before leaving the commingling centre (day −1), upon arrival at the fattening unit (day 0), and 7 days post-arrival (day 7). These bulls were part of a larger study aimed at testing whether the animals have some benefit from the supplementation of live yeast and selenium through slow-release rumen boluses that were administered to half of them before departure from France (‘Yeast’ vs ‘Control’ group). The effect of the supplementation on the parameters considered in this study was included in the statistical analysis to account for the structure of the experimental design. Transport affected the initial body weight of the animals, which dropped on day 0 and it was not fully recovered on day 7. Most plasma traits of protein, energy, hepato-muscle, and mineral profiles were affected by transport. Cortisol was also assessed and peaked at day 0. The footprint of the long-distance transport on bulls’ metabolic profile indicated a combination of stress, energy deficit, and muscle damage, with a certain degree of dehydration and liver impairment. Plasma traits measured on day −1 highlighted that stressful conditions and physiological responses of the bulls to recover homeostasis already started during the commingling phase before departure. No effect of supplementation was detected, except for higher selenium plasma level in Yeast bulls at day 7. Among blood parameters, non-esterified fatty acids, total protein, cortisol, glucose, and iron were those responsible for most of the variation in metabolic profile of bulls undergoing long journey. Therefore, these traits might be used as major biomarkers to assess stress in transported beef cattle, helping to identify critical situations for which proper mitigating actions should be taken. The outcomes of this study suggested that preventive measures against transport stress in beef cattle should start at the commingling of the animals in the collection centers, thus before departure.