Industry 5.0 paradigm has renewed interest in the human sphere, emphasizing the importance of workers’ well-being in manufacturing activities. In such context, collaborative robotics originated as a technology to support humans in tiring and repetitive tasks. This study investigates the effects of assembly complexity in Human-Robot collaboration using physiological indicators of cognitive effort. In a series of experiments, participants performed assembly processes of different products with varying complexity, in two modalities: manually and with cobot assistance. Physiological measures, including skin conductance, heart rate variability and eye-tracking metrics were collected. The analysis of physiological signals showed trends suggesting the impact of assembly complexity and cobot support. One key finding of the study is that a single physiological signal usually may not provide a complete understanding of cognitive load. Therefore, a holistic approach should be followed. This approach highlighted the importance of considering multiple measures simultaneously to accurately assess workers’ well-being in industrial environments.