Background: With the aim of integrating simulation training into the training of perfusionists, we examined whether the participants were able to transfer a specific learning content to the same and different situations and assessed their feedback on the simulation training. Eye-tracking was tested as a measure and supplemented by additional measures. Methods: A 2 × 2 mixed design was used, with test time (pre- and post-test) and training group (same and different content training) as factors. In the pre- and post-test, the participant had to handle a critical situation on the cardiopulmonary bypass, namely, a drop in arterial partial oxygen pressure. Between the two test times, the participant practised under guidance the handling of either the same critical situation (Group 1) or a different one, that is, impaired venous return (Group 2). Dependent measures were fixations of the eyes on specific areas of interest on the heart-lung machine, measures of latency and subjective assessments. Moreover, participants gave feedback on the simulation training. Results: Fixation analyses showed that the training led to an increased gaze on areas of interest relevant to the drop in arterial partial oxygen pressure in both groups, with a significant increase only for Group 1. The surveys revealed a great interest in the integration of simulation training into education. Conclusion: In combination with other measures, eye-tracking is suitable for the evaluation of simulation training. Due to the positive training effects and positive participant feedback, the integration of simulation into the training of perfusionists is advocated. Concerning transfer of learning content, more research is needed.