Humane alternatives to harmful educational animal use include ethically-sourced cadavers, models, mannequins, mechanical simulators, videos, computer and virtual reality simulations, and supervised clinical and surgical experiences. In many life and health sciences courses, however, traditional animal use persists, often due to uncertainty about the educational efficacy of humane alternatives. The most recent comprehensive reviews assessing learning outcomes of humane teaching methods, in comparison to harmful animal use, were published more than 10 years ago. Therefore, we aimed to collate and analyse the combined evidence from recent and older studies about the efficacy of humane teaching methods. Using specific search terms, we systematically searched the Web of Science, SCOPUS, and EMBASE databases for relevant educational studies. We extracted information on publication years, the country in which the study was conducted, field, humane teaching methods, form of learning outcome assessment, and the learning outcome of the humane teaching methods, in comparison with harmful animal use. We found 50 relevant studies published from 1968-2020, primarily stemming from the USA, UK, and Canada. Humane teaching methods produced learning outcomes superior (30%), equivalent (60%), or inferior (10%) to those produced by traditional harmful animal use. In conclusion, a wide-spread implementation of humane teaching methods would not only preserve learning outcomes, but may in fact be beneficial for animals, students, educators, and institutions.