A Londoner by birth, Starling studied medicine at Guy's Hospital, London, where he obtained his MB in 1889 and eventually became head of the department of physiology. In 1899 he moved to University College, London, to become Jodrell Professor of Physiology, a position he held until his death.

In 1896 Starling introduced the concept of the Starling equilibrium, which tried to relate the pressure of the blood to its behavior in the capillary system. He realized that the high pressure of the arterial system is enough to force fluids through the thin-walled capillaries into the tissues. But as the blood is divided through more and more capillaries its pressure falls. By the time it reaches the venous system the pressure of the fluid in the surrounding tissues is higher than that of blood in the venous capillaries, allowing much of the fluid lost from the arterial side to be regained. In theory the two systems should be in a state of equilibrium. In reality the system is complicated by the hydrostatic pressure of the blood and the osmotic pressure arising from the various salts and proteins dissolved in it.

In 1915 Starling formulated the important law (Starling's law) stating that the energy of contraction of the heart is a function of the length of the muscle fiber. As the heart fills with blood the muscle is forced to expand and stretch; the force with which the muscle contracts to expel the blood from the heart is simply a function of the extent to which it has been stretched. The curve that relates the two variables of the heart – pressure and volume – is known as Starling's curve.

Starling's best-known work was his collaboration with William Bayliss in the discovery of the hormone secretin in 1902. The normal pancreas releases a number of juices into the duodenum to aid in the process of digestion. By cutting all the pancreatic nerves and noting the continuing secretion of the pancreatic juices Starling and Bayliss showed that the release of the juices was not under nervous control. They concluded that a chemical, rather than a nervous, message must be sent to the pancreas through the blood when food enters the duodenum. They proposed to call the chemical messenger secretin. For the general class of such chemicals Starling proposed, in 1905, the term ‘hormone’, from the Greek root meaning to excite. Thus endocrinology – a major branch of medicine and physiology – had been created.

It was widely known before the outbreak of World War I that Starling and Bayliss had been the strongest of the candidates for the Nobel Prize for physiology and medicine. However, as no awards were made during the war they missed out completely; the prizes after 1918 were awarded for more recent work. As for honors from his own country, Starling was far too outspoken about the incompetent direction of the war even to be considered.