CONTEXT Three-dimensional (3-D) visualisation in anatomical education has been shown to be broadly beneficial for students. However, there is limited research on the relative efficacy of 3-D modalities. This study compares knowledge performance, mental effort and instructional efficiency between autostereoscopic 3-D visualisation (holograms), monoscopic 3-D visualisation (3-DPDFs) and a control (2-D printed images). METHODS A cardiac anatomy model was used to generate holograms, 3-DPDFs and 2-D printed images. Nursing student participants (n = 179) were randomised into three groups: holograms (n = 60), 3-DPDFs (n = 60) and printed images (n = 59). Participants completed a pre-test followed by a self-study period using the anatomical visualisation. Afterwards, participants completed the NASA-Task Load Index (NASA-TLX) cognitive load instrument and a knowledge post-test. RESULTS Post-test results showed participants studying with holograms (median = 80.0, interquartile range [IQR] = 66.7-86.7) performed significantly better regarding cardiac anatomy knowledge than participants using 3-DPDF (median = 66.7, IQR = 53.3-80.0, p = 0.008) or printed images (median = 66.7, IQR = 53.3-80.0, p = 0.007). Mental effort scores, on a scale from 1 to 20, showed hologram (mean = 4.9, standard deviation [SD] = 3.56) and 3-DPDF participants (mean = 4.9, SD = 3.79) reported significantly lower cognitive load than printed images (mean = 7.5, SD = 4.9, p < 0.005). Instructional efficiency (E) of holograms (E = 0.35) was significantly higher than printed images (E = -0.36, p < 0.001), although not significantly higher than 3-DPDF (E = 0.03, p = 0.097). CONCLUSIONS Participants using holograms demonstrated significant knowledge improvement over printed images and monoscopic 3-DPDF models, suggesting additional depth cues from holographic visualisation provide benefit in understanding spatial anatomy. Mental effort scores and instructional efficiency of holograms indicate holograms are a cognitively efficient instructional medium. These findings highlight the need for further study of novel 3-D technologies and learning performance.