Galenism's final defeat came from a combination of the negativism of Paracelsus and the constructivism of the Italian Renaissance anatomists, such as Vesalius in the 16th century. In the 1530s, the Flemish Anatomist and physician Andreas Vesalius took on a project to translate many of Galen's Greek texts into Latin. Vesalius' most famous work, De humani corporis fabrica, was greatly influenced by Galenic writing and form. Seeking to examine critically Galen's methods and outlook, Vesalius turned to human cadaver dissection as a means of verification. Galen's writings were shown by Vesalius to describe details present in monkeys but not in humans, and he demonstrated Galen's limitations through books and hands-on demonstrations despite fierce opposition from orthodox pro-Galenists such as Jacobus Sylvius. Since Galen states that he is using observations of monkeys (human dissection was prohibited) to give an account of what the body looks like, Vesalius could portray himself as using Galen's approach of description of direct observation to create a record of the exact details of the human body, since he worked in a time when human dissection was allowed. Galen argued that monkey anatomy was close enough to humans for Physicians to learn anatomy with monkey dissections and then make observations of similar structures in the wounds of their patients, rather than trying to learn anatomy only from wounds in human patients, as would be done by students trained in the Empiricist model. The examinations of Vesalius also disproved medical theories of Aristotle and Mondino de Liuzzi. One of the best known examples of Vesalius' overturning of Galenism was his demonstration that the interventricular septum of the heart was not permeable, as Galen had taught (Nat Fac III xv). However, this had been revealed two years before by Michael Servetus in his fateful "Christianismi restitutio" (1553) with only three copies of the book surviving, but these remaining hidden for decades; the rest were burned shortly after its publication because of persecution of Servetus by religious authorities.

Michael Servetus, using the name "Michel de Villeneuve" during his stay in France, was Vesalius' fellow student and the best Galenist at the University of Paris, according to Johann Winter von Andernach, who taught both. In the Galenism of the Renaissance, editions of the Opera Omnia by Galen were very important. It was begun in Venice in 1541–1542 by the Guinta. There were fourteen editions of the book from that date until 1625. Just one edition was produced from Lyon between 1548 and 1551. The Lyon edition has commentaries on breathing and blood streaming that correct the work of earlier renowned authors such as Vesalius, Caius or Janus Cornarius. "Michel De Villeneuve" had contracts with Jean Frellon for that work, and the Servetus scholar-researcher Francisco Javier González Echeverría presented research that became an accepted communication in the International Society for the History of Medicine, which concluded that Michael De Villeneuve (Michael Servetus) is the author of the commentaries of this edition of Frellon, in Lyon.

Various attempts have been made to classify Galen's vast output. For instance Coxe (1846) lists a Prolegomena, or introductory books, followed by 7 classes of treatise embracing Physiology (28 vols.), Hygiene (12), Aetiology (19), Semeiotics (14), Pharmacy (10), Blood letting (4) and Therapeutics (17), in addition to 4 of aphorisms, and spurious works. The most complete compendium of Galen's writings, surpassing even modern projects like the Corpus Medicorum Graecorum, is the one compiled and translated by Karl Gottlob Kühn of Leipzig between 1821 and 1833. This collection consists of 122 of Galen's treatises, translated from the original Greek into Latin (the text is presented in both languages). Over 20,000 pages in length, it is divided into 22 volumes, with 676 index pages. Many of Galen's works are included in the Thesaurus Linguae Graecae, a digital library of Greek literature started in 1972. Another useful modern source is the French Bibliothèque interuniversitaire de médecine (BIUM).

Galen was the physician to Commodus for much of the emperor’s life and treated his Common illnesses. According to Dio Cassius 72.14.3–4, in about 189, under Commodus’ reign, a pestilence occurred which at its height killed 2,000 people a day in Rome. This was most likely the same plague that struck Rome during Marcus Aurelius’ reign.

After the collapse of the Western Empire the study of Galen and other Greek works almost disappeared in the Latin West. In contrast, in the predominantly Greek-speaking eastern half of the Roman empire (Byzantium), many commentators of the subsequent centuries, such as Oribasius, physician to the Emperor Julian who compiled a Synopsis in the 4th century, preserved and disseminated Galen's works, making Galenism more accessible. Nutton refers to these authors as the "medical refrigerators of antiquity". In late antiquity, medical writing veered increasingly in the direction of the theoretical at the expense of the practical, with many authors merely debating Galenism. Magnus of Nisibis was a pure theorist, as were John of Alexandria and Agnellus of Ravenna with their lectures on Galen's De Sectis. So strong was Galenism that other authors such as Hippocrates began to be seen through a Galenic lens, while his opponents became marginalised and other medical sects such as Asclepiadism slowly disappeared. Greek Medicine was part of Greek culture, and Syrian Eastern Christians came in contact with it while the Eastern Roman Empire (Byzantium) ruled Syria and Western Mesopotamia, regions that were conquered from Byzantium in the 7th century by Arab Muslims. After AD 750, Muslims had these Syrian Christians make the first translations of Galen into Arabic. From then on, Galen and the Greek medical tradition in general became assimilated into the medieval and early modern Islamic Middle East.

From the 11th century onwards, Latin translations of Islamic medical texts began to appear in the West, alongside the Salerno school of thought, and were soon incorporated into the curriculum at the universities of Naples and Montpellier. From that time, Galenism took on a new, unquestioned authority, Galen even being referred to as the "Medical Pope of the Middle Ages". Constantine the African was amongst those who translated both Hippocrates and Galen from Arabic. In addition to the more numerous translations of Arabic texts in this period, there were a few translations of Galenic works directly from the Greek, such as Burgundio of Pisa's translation of De complexionibus. Galen's works on anatomy and Medicine became the mainstay of the medieval physician's university curriculum, alongside Ibn Sina's The Canon of Medicine, which elaborated on Galen's works. Unlike pagan Rome, Christian Europe did not exercise a universal prohibition of the dissection and autopsy of the human body and such examinations were carried out regularly from at least the 13th century. However, Galen's influence was so great that when dissections discovered anomalies compared with Galen's anatomy, the Physicians often tried to fit these into the Galenic system. An Example of this is Mondino de Liuzzi, who describes rudimentary blood circulation in his writings but still asserts that the left ventricle should contain air. Some cited these changes as proof that human anatomy had changed since the time of Galen.

Galen's approach to Medicine became and remains influential in the Islamic world. The first major translator of Galen into Arabic was the Syrian Christian Hunayn ibn Ishaq. He translated (c. 830–870) 129 works of "Jalinos" into Arabic. Arabic sources, such as Rhazes (Muhammad ibn Zakarīya Rāzi AD 865–925), continue to be the source of discovery of new or relatively inaccessible Galenic writings. One of Hunayn's Arabic translations, Kitab ila Aglooqan fi Shifa al Amrad, which is extant in the Library of Ibn Sina Academy of Medieval Medicine & Sciences, is regarded as a masterpiece of Galen's literary works. A part of the Alexandrian compendium of Galen's work, this 10th-century manuscript comprises two parts that include details regarding various types of fevers (Humyat) and different inflammatory conditions of the body. More important is that it includes details of more than 150 single and compound formulations of both herbal and animal origin. The book provides an insight into understanding the traditions and methods of treatment in the Greek and Roman eras. In addition, this book provides a direct source for the study of more than 150 single and compound drugs used during the Greco-Roman period.

In his time, Galen's reputation as both physician and Philosopher was legendary, the Emperor Marcus Aurelius describing him as "Primum sane medicorum esse, philosophorum autem solum" (first among doctors and unique among Philosophers Praen 14: 660). Other contemporary authors in the Greek world confirm this including Theodotus the Shoemaker, Athenaeus and Alexander of Aphrodisias. The 7th-century poet George of Pisida went so far as to refer to Christ as a second and neglected Galen. Galen continued to exert an important influence over the theory and practice of Medicine until the mid-17th century in the Byzantine and Arabic worlds and Europe. Hippocrates and Galen form important landmarks of 600 years of Greek Medicine. A. J. Brock describes them as representing the foundation and apex respectively. A few centuries after Galen, Palladius Iatrosophista stated, in his commentary on Hippocrates, that Hippocrates sowed and Galen reaped.

Galen's principal interest was in human anatomy, but Roman law had prohibited the dissection of human cadavers since about 150 BC. Because of this restriction, Galen performed anatomical dissections on living (vivisection) and dead animals, mostly focusing on pigs and primates. This work was useful because Galen believed that the anatomical structures of these animals closely mirrored those of humans. Galen clarified the anatomy of the trachea and was the first to demonstrate that the larynx generates the voice. In one experiment, Galen used bellows to inflate the lungs of a dead animal. Galen's work on the anatomy remained largely unsurpassed and unchallenged up until the 16th century in Europe. In the middle of the 16th century, the Anatomist Andreas Vesalius challenged the anatomical knowledge of Galen by conducting dissections on human cadavers. These investigations allowed Vesalius to refute aspects of Galen's anatomy.

Another convincing case where understanding of the body was extended beyond where Galen had left it came from these demonstrations of the nature of human circulation and the subsequent work of Andrea Cesalpino, Fabricio of Acquapendente and william Harvey. Some Galenic teaching, such as his emphasis on bloodletting as a remedy for many ailments, however, remained influential until well into the 19th century.