Over the years, much criticism against animal use for physiology teaching has been made. Hence, replacement by suitable alternatives has increased in several pedagogical approaches. This study examined students' perceptions of animal versus virtual (video/computer) laboratory classes in physiological sciences associated with the effectiveness of the problem-based learning (PBL) hybrid curriculum. Three cohorts of medical students from the University of Ribeirão Preto, who participated in animal or virtual physiology classes or both, were asked to fill out a 5-point Likert questionnaire about knowledge acquisition/motivation, importance to PBL learning goals, skills acquired, need for animal use, academic formation, learning impairment, and alternative methods. We also assessed their grades in the final exam. A total of 350 students were included, in which 108 participated only in virtual classes, 120 only in practical animal laboratory classes, and 122 in both approaches. The majority agreed that the two methods improved their knowledge acquisition/motivation and helped to reinforce tutorial goals and to acquire skills. However, the cohort who experienced both approaches favored animal laboratory. Students believe animal use is needed and did not impair their learning. Conversely, their opinion about academic formation without animal laboratory classes was divided, as was whether this approach inspired them to seek alternative methods. Despite the different perceptions, there was no difference among the groups' final grades (7.3 ± 1 vs. 7.2 ± 1 vs. 7.2 ± 2 for virtual or practical animal laboratory classes or both, respectively). Therefore, virtual activities are not as effective as animal use in the opinions of the students, but they are successful strategies in physiology learning that can be used in practical classes in a hybrid PBL curriculum.