Her studies included mathematics, Renaissance classics, the languages of Latin, Spanish and French besides her native Portuguese, etiquette, and Christian doctrine.

Their marriage would bring a strong alliance between Spain and Portugal, in accordance with the wishes of their grandparents, Isabella I of Castille and Ferdinand II of Aragón.

Many in Portugal took their Infanta's rejection as an offense, but Isabella remained determined she would marry her powerful cousin or enter a convent.

There were many more advantages – she was closer to him in age (she was only 3 years his junior), fluent in Spanish, and offered a dowry of 900,000 Portuguese cruzados (or Castilian folds), which was more than enough to solve many of his financial problems brought on by the Italian War of 1521–26.

Charles intended to wed and then leave his future wife as regent to govern Spain while he went to Central Europe to deal with political and religious troubles there.

As Charles had planned, he appointed Isabella regent of Spain during his absence from the peninsula to lead his military campaigns and attend the administration of his other kingdoms between 1529–33 and 1537–39.

[citation needed] With both civil and criminal jurisdiction, Isabella appointed Diego López de Medrano as her mayordomo mayor (High Steward), entrusting him with the organization and governance of the royal palace.

[citation needed] Isabella effectively defended the royal power in order to ensure the monarch's authority, as a response towards the previous rebellions against Charles for his foreign relationships.

In foreign policy, Isabella actively intervened in the negotiations of marital alliances between the French and Spanish royal families.

There is speculation that she suffered from consumption, with a contemporary describing her: "The Empress is the greatest pity in the world, she is so thin that she does not resemble a person".

In 1539, she became pregnant for the seventh time, but contracted another fever in the third month that caused antenatal complications and gave birth to a stillborn son.

[12] Charles was left so devastated that he could not bring himself to accompany his wife's body to the Royal Chapel of Granada, the burial place of the Catholic Monarchs.

Decomposition had so disfigured Isabella's body, however, that Gandía could not recognize her and was allegedly so horrified at what death had done to her beauty that he later became a Jesuit, gaining fame as San Francisco de Borja.

Crecquillon composed his Missa Mort m'a privé as a memorial to her, which expresses her husband's grief and wish for a heavenly reunion with his beloved wife.

[16] Isabella had seven children with Charles, of whom three survived including King Philip II of Spain and Maria, another Holy Roman Empress.

[citation needed] Isabella of Portugal is portrayed by Blanca Suárez in the TVE series Carlos, Rey Emperador.