The purpose of this study was to analyze the influence of pain and no pain expectation on pupil diameter and the cognitive assessment of pain using pupillometry and the visual analog scale (VAS). This was an experimental cross-sectional study in which painful stimulation of the muscle masseter was produced by palpation with an algometer in a sample of 30 healthy participants. Before the painful stimulation, pain expectation and no pain expectation situations were induced by employing instructional videos. Cognitive and physiological pain responses were measured by the VAS and by pupillometry. Visual analog scale score was assessed at the end of each experimental condition, and pupillometry measurements were recorded at different moments of the experimental process (events). The pain score and larger pupil diameter for all events (except, as expected, for the initial baseline), were statistically significant for the pain expectation condition compared with the no pain expectation. Within the conditions of this study, pain expectation modulated both cognitive assessment of pain (VAS) and pupil diameter. These findings suggest that pupil diameter may be a useful and complementary tool with the VAS for pain assessment. Furthermore, the findings support models postulating that emotion and cognition are important aspects involved in pain perception, which is in line with the recent International Association for the Study of Pain definition of pain.