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Contemporary research demonstrated that undergraduate medical students rated an augmented reality learning tool entitled "human muscular arm avatar" as useful for their self-directed individual learning. Beyond this subjective assessment, it was largely unknown whether avatar-based study would also improve knowledge test scores at an objective level. This study aimed to investigate knowledge gain and student perception following a short constructively aligned cooperative learning approach, utilizing innovative technology and classical prosection-based learning. In a randomized, quantitative pre-post study design, experienced medical students studied functional anatomy of three muscles with either the avatar or prosected human specimens. Ninety-six students completed mock examinations and surveys on their learning experience. Descriptive and internal reliability analyses were performed along with effect size calculation and item analyses. Learning satisfaction and item-specific knowledge gain were observed following both interventions. Female students demonstrated, respectively, medium or large learning gains after their learning with the virtual avatar or prosected human specimen. Male students yielded minor learning gain with the virtual avatar and large learning gain with prosections. Gender- and intervention-related differences in perceived learning gain, interest, and embodied experience were observed. The integration of constructive alignment and cooperative learning with virtual avatars and prosected human specimens supported experienced students in learning functional anatomy of specific muscles. Gender- and intervention-related differences in perception may have influenced item-specific knowledge gain. Mentoring, one-to-one instruction, practical assessments, and technological optimization could further enhance the learning experience.