Background: Road transport companies utilize transport capacities as fixed compositions of tractors and semi-trailers, while the possibility of exchanging semi-trailers is considered ad hoc, after some unforeseen circumstances emerge on the route. Such an approach is a limiting factor in achieving optimal utilization of transport capacities, and consequently affects profitability. We proposed a new concept of vehicle fleet management where semi-trailers would not be permanently assigned to the tractors, but could be dynamically reassigned, to utilize optimally the vehicle fleet, on considering the planned itinerary, the driver’s working hours, and traffic conditions. Methods: We set the key performance indicators and developed a prototype application based on the concept of dynamic semi-trailer allocation. We simulated the use of the prototype application on the historical data collected in the case study on one of the leading transport companies in the cold chain, and evaluated the benefits that may be achieved thereof. Results: Simulation showed that implementing the proposed concept enabled the reduction of the vehicle workdays spent on-the-route, the reduction of the number of vehicle compositions needed to handle the transport demand, and improved the quality of service. Conclusions: The proposed concept is beneficial for transport companies that operate large vehicle fleets on long-haul routes, with several transport orders per route. The prototype application may be the basis for developing a fully functional application that can be integrated into the fleet management system.