The earliest trace of Leonardo's interest in anatomy is in his painting of Saint Jerome, which dates from his youth. The neck and shoulders already reveal some knowledge of muscle anatomy. While contemporary artists like Michelangelo confined their studies to superficial anatomy, Leonardo extended his research to the deeper parts of the body. The studies of a horse's entrails and the spinal chord of a frog prove that in this phase he resorted to animal dissection. In one series of drawings, he analyzed the internal dimensions of the skull. He also sought to locate the psychic faculties inside three circular intracerebral cavities. Leonardo espoused the traditional view of nerves as tubes carrying air flows that cause muscles to contract by inflation.

Leonardo resumed his anatomical studies in about 1510, after a break of nearly a decade. His studies in mechanics influenced his later anatomical work. He now analyzed the body's articulations as semi-articulated joints governed by the laws of the lever. The routine practice of dissection exposed him to the awesome complexity of anatomical data. He was convinced that every anatomical structure has a precise function. No detail must therefore be overlooked in the visual representation. He resorted to innovative graphic devices, such as "see-through" images, exploded views, drawings of the body from different vantage points, and the depiction of muscles as lines of force.

Even Leonardo's studies in hydrodynamics powerfully influenced his anatomical research. He examined cardiocirculatory, respiratory, and urogenital systems as networks of channels carrying fluids in motion. The passage of blood through the cardiac valves creates vortexes similar to those generated by water in a narrow channel. Conversely, Leonardo draws an analogy between underground streams and the veins of the human body. The water carried by the streams brings nutrients to the Earth's body, as blood does to the human body. The fetus grows immersed in the amniotic fluid as the Earth's continents grow by emerging from the surrounding seas. Leonardo likens the principles that regulate the ramifications of blood vessels to the rules governing the branching of trees. The heart generates vessels as seeds engender plants.