In the Renaissance, greater attention to experimentation in the various disciplines of knowledge resulted, in the medical field, in the further development of anatomical studies. Near the end of the 15th century Antonio Benivieni, a physician in the Florentine Hospital di Santa Maria Nuova, made over one hundred clinical observations, through careful post-mortem examinations, for which he is commonly considered the forerunner of pathological anatomy.

During this same period, progress in anatomical studies proceeded at the same rhythm as the publishing of works dedicated to pharmacology and therapeutics. In 1498 the first work of pharmocopeia, the Nuovo Receptario, was published in Florence.

In the early years of the 16th century Leonardo da Vinci devoted himself to studying the human body, practicing the dissection of cadavers in the Florentine Hospital of Santa Maria Nuova. Of this activity there remain numerous drawings, justly considered masterpieces of scientific anatomy. His observations, focussed on comparison between the human body and the machine, revolutionised traditional medical science. Leonardo was the first to represent segments of the skeleton and to use the example of the lever to explain how the joints worked. He represented the muscular fascia by wires or cords, revealing their mechanical action. In his studies on the cervical vertebrae, he compared the muscles that support the neck to the shrouds that hold up the mast in a ship.

In 16th century Tuscany, surgery was taught not only at the University of Pisa but also in various schools of surgery annexed to hospitals, such as that of the Ceppo Hospital in Pistoia and the School of Medicine and Surgery at the Santa Maria Nuova Hospital in Florence. And it was in the Florence hospital that, at the order of Cosimo I de' Medici, all of the physicians and surgeons in Tuscany had to take an examination qualifying them for the medical profession. It was during those years that surgery, thanks also to the fundamental contribution of Andrea Vesalio in the field of anatomical-physiological studies (De humani corporis fabrica, 1543), came to be considered no longer an inferior craft left almost exclusively to barbers, but a subject to be included in the curriculum of a doctor.

Within the sphere of the Galilean Accademia del Cimento (1657-1667), the Florentine alcohol thermometer was developed, starting from the thermoscope. This instrument was employed not only in meteorology but also in the medical field. A variation of the instrument, the so-called "frog thermometer", was used to measure the temperature of the human body (the Academy’s thermometers are now displayed at the Institute and Museum of the History of Science in Florence). The members of the Academy also carried out their activity using a crucially important instrument, the microscope, invented in the early 17th century.

A decisive step in the development of medicine, especially as regards experimental research, is represented by the ingenious experiments of Francesco Redi, a physician from Arezzo who, through an exemplary experimental procedure, demonstrated the falsity of the belief in spontaneous generation. Another member of the Cimento, Giovanni Alfonso Borelli, conducted very important studies on the bone structure of animals in relation to their weight and movement. Particularly significant were the studies of Lorenzo Bellini, a pupil of Borelli, who in 1653 explained how, in the kidneys, the blood is separated from what he called the "superfluous serosity", that is, the urine.

Observations on the circulation of the blood, which had begun with the English physiologist William Harvey, who had identified the heart as the pump that mechanically regulates the dynamics of the circulatory motion, were conducted in Tuscany in the 17th century by Marcello Malpighi. In 1661, utilising the microscope, Malpighi proved the existence of the capillaries that serve as connection between the venous and the arterial vascular systems. And it was in Tuscany that the first experiments in blood transfusion were made. In 1654 Francesco Folli da Poppi, inspired by his own experiments in grafting plants, performed blood transfusions, believing that they could cure diseases and fortify persons of weak constitution.

In the 18th century Giovan Battista Morgagni, one of the outstanding figures in the field of medical studies, established a causal relationship between diseases and anatomical lesions in the organs affected by them, observations that still today merit him the title of founder of Pathological Anatomy. In Tuscany, anatomical studies were developed also through the work of Abbot Felice Fontana. In the Museo di Fisica e Storia Naturale, he set up a waxworks shop for the reproduction of anatomical parts to be used in teaching, collaborating with famous wax-modellers in preparing the anatomical wax figures displayed today in the Florentine Museum of "La Specola".

In 1756 Tuscany was swept by a violent epidemic of smallpox. The grand-ducal physician Giovanni Targioni Tozzetti inoculated six orphans with the cowpox virus. This practice, only recently experimented, did not meet with the unanimous approval of the scientific world. The enlightened reformist policy of the Lorraines brought Tuscany to the forefront in the affirmation of this type of prevention. Grand Duke Peter Leopold of Lorraine himself underwent vaccination at the advice of his mother, the Austrian Empress Maria Teresa, who had contracted the disease in 1767. The Grand Duke was vaccinated by the physician Angelo Gatti at the Villa of Poggio Imperiale at 11 o'clock on the morning of May 13, 1769.

During the last years of the 18th century, Tuscany accepted the premises for the constitution of psychiatry as a discipline with a status of its own. Vincenzo Chiarugi, in his crucially important work Della pazzia in genere e in specie (On madness in general and in its specific types) considered mental illnesses on the same level as other pathologies, and believed that the mentally ill should be treated in special facilities and not segregated as had been the custom up to then. For this purpose, the Bonifazio Hospital in Florence was delegated to accommodate psychiatric patients coming from the now inadequate Santa Dorotea Hospital which, towards the end of the 18th century, had been the model for the new Psychiatric Hospital of Lucca. In the 19th century the new sensitivity toward pathologies of this kind led to the founding and reorganising of other hospitals, built to satisfy the new requisites; some examples are the former Psychiatric Hospital of San Niccolò in Siena, the former Psychiatric Hospital of San Salvi in Florence and the former Psychiatric Hospital of Arezzo.

Important innovations took place also in the field of obstetrics. Giuseppe Vespa, who had studied at the Paris School of Obstetrics, began to teach at the Hospital di Santa Maria Nuova in Florence toward the middle of the 18th century, just a few months after the first Italian Chair of Obstetrics had been instituted at Bologna. Thanks to him, Local Obstetrical Units were set up in every quarter of the city, each with a midwife and a surgeon to be called in when necessary. Each surgeon was assisted by two of the best students in surgery. The Local Units thus served an educational purpose as well as providing important centres for the organisation of emergencies.

The finest 19th-century work on anatomy was written by Paolo Mascagni, who conducted a fundamentally important study of the lymphatic vessels. In 1823, seven years after his death, the Anatomia universa, the monumental work to which the scientist had devoted himself for years, was published in Pisa.

The 19th century marked a decisive moment in the sphere of medical-biological studies, with the definition of questions such as reproduction, the determination of sex and hereditary traits. In Tuscany the activity of Giovanni Battista Amici, a scientist from Modena, was highly important. His innovations in the field of microscope construction provided researchers in anatomy with efficacious instruments for studying the structure of minute bodies. The Pistoian Filippo Pacini, the first professor in Italy to teach a course in Microscopic Anatomy, discovered the tactile corpuscles in 1853, while still a student, described the structure of the retina and, a forerunner in the field of bacteriology, described the cholera vibrio. He was, together with Pietro Betti and Maurizio Bufalini, one of the founders of the Florentine Physical Medicine Society, at which the Pathological Museum was set up.

The complex history of medicine in Tuscany can be evoked in part starting from the ancient Hospital of Santa Maria della Scala, within whose circle of influence lay numerous fortified farms, such as the Cuna Grange and the Serre Grange which, in addition to providing for the subsistence of the main centre, offered lodging and assistance to travellers. Among these relief and charity institutions were some of the greatest historic hospitals in Tuscany (the Misericordia e Dolce Hospital in Prato, the Ceppo Hospital in Pistoia, the Santa Chiara Hospital in Pisa, the Hospital "degli Innocenti" and that of San Giovanni di Dio in Florence), as well as numerous Misericordia Confraternities, an eloquent example of which is provided by the Museum of the Confraternity in Anghiari. Noteworthy as regards the history of medicine are, in addition to the various thermal stations, the numerous historic pharmacies scattered over the territory; among them, special mention is due the "Officina Profumo Farmaceutica di Santa Maria Novella" in Florence, the "Spezieria di Santa Fina" in San Gimignano, the pharmacy of the Archi-coenoby of Monte Oliveto Maggiore, the pharmacy of the Camaldoli Monastery, the pharmacy of the La Verna Sanctuary, the "Spezieria dello Spedale Serristori" in Figline Valdarno and the pharmacy of the "Certosa" at Calci.