The adoption of Digital Twin (DT) solutions for industrial purposes is increasing among small- and medium-sized enterprises and is already being integrated into many large-scale companies. As there is an increasing need for faster production and shortening of the learning curve for new emerging technologies, Virtual Reality (VR) interfaces for enterprise manufacturing DTs seem to be a good solution. Furthermore, with the emergence of Industry 5.0 (I5.0) paradigm, human operators will be increasingly integrated in the systems interfaces though advanced interactions, pervasive sensors, real time tracking and data acquisition. This scenario is especially relevant in collaborative automated systems where the introduction of immersive VR interfaces based on production cell DTs might provide a solution for the integration of the human factors in the modern industrial scenarios. This study presents experimental results of the comparison between users controlling a physical industrial robot system via a traditional teach pendant and a DT leveraging a VR user interface. The study group involves forty subjects including experts in robotics and VR as well as non-experts. An analysis of the data gathered in both the real and the virtual use case scenario is provided. The collected information includes time for performing a task with an industrial robot, stress level evaluation, physical and mental effort, and the human subjects’ perceptions of the physical and simulated robots. Additionally, operator gazes were tracked in the VR environment. In this study, VR interfaces in the DT representation are exploited to gather user centered metrics and validate efficiency and safety standards for modern collaborative industrial systems in I5.0. The goal is to evaluate how the operators perceive and respond to the virtual robot and user interface while interacting with them and detect if any degradation of user experience and task efficiency exists compared to the real robot interfaces. Results demonstrate that the use of DT VR interfaces is comparable to traditional tech pendants for the given task and might be a valuable substitute of physical interfaces. Despite improving the overall task performance and considering the higher stress levels detected while using the DT VR interface, further studies are necessary to provide a clearer validation of both interfaces and user impact assessment methods.