Helmholtz continued to work for several decades on several editions of the handbook, frequently updating his work because of his dispute with Ewald Hering who held opposite views on spatial and color vision. This dispute divided the discipline of physiology during the second half of the 1800s.

Drawing on the earlier work of Sadi Carnot, Benoît Paul Émile Clapeyron and James Prescott Joule, he postulated a relationship between mechanics, heat, light, electricity and magnetism by treating them all as manifestations of a single force (energy in modern terms). He published his theories in his book Über die Erhaltung der Kraft (On the Conservation of Force, 1847).

Helmholtz's first academic position was as a Teacher of Anatomy at the Academy of Arts in Berlin in 1848. He then moved to take a post of associate professor of physiology at the Prussian University of Königsberg, where he was appointed in 1849. In 1855 he accepted a full professorship of anatomy and physiology at the University of Bonn. He was not particularly happy in Bonn, however, and three years later he transferred to the University of Heidelberg, in Baden, where he served as professor of physiology. In 1871 he accepted his final university position, as professor of physics at the University of Berlin.

In the 1850s and 60s, building on the publications of william Thomson, Helmholtz and william Rankine popularized the idea of the heat death of the universe.

In 1851, Helmholtz revolutionized the field of ophthalmology with the invention of the ophthalmoscope; an instrument used to examine the inside of the human eye. This made him world-famous overnight. Helmholtz's interests at that time were increasingly focused on the physiology of the senses. His main publication, titled Handbuch der Physiologischen Optik (Handbook of Physiological Optics or Treatise on Physiological Optics), provided empirical theories on depth perception, color vision, and motion perception, and became the fundamental reference work in his field during the second half of the nineteenth century. In the third and final volume, published in 1867, Helmholtz described the importance of unconscious inferences for perception. The Handbuch was first translated into English under the editorship of James P. C. Southall on behalf of the Optical Society of America in 1924-5. His theory of accommodation went unchallenged until the final decade of the 20th century.

In 1863, Helmholtz published Sensations of Tone, once again demonstrating his interest in the physics of perception. This book influenced musicologists into the twentieth century. Helmholtz invented the Helmholtz resonator to identify the various frequencies or pitches of the pure sine wave components of complex sounds containing multiple tones.

Other students and research associates of Helmholtz at Berlin included Max Planck, Heinrich Kayser, Eugen Goldstein, Wilhelm Wien, Arthur König, Henry Augustus Rowland, Albert A. Michelson, Wilhelm Wundt, Fernando Sanford and Michael I. Pupin. Leo Koenigsberger, who was his colleague 1869–1871 in Heidelberg, wrote the definitive biography of him in 1902.

In 1871, Helmholtz moved from Heidelberg to Berlin to become a professor in physics. He became interested in electromagnetism and the Helmholtz equation is named for him. Although he did not make major contributions to this field, his student Heinrich Rudolf Hertz became famous as the first to demonstrate electromagnetic radiation. Oliver Heaviside criticised Helmholtz's electromagnetic theory because it allowed the existence of longitudinal waves. Based on work on Maxwell's equations, Heaviside pronounced that longitudinal waves could not exist in a vacuum or a homogeneous medium. Heaviside did not note, however, that longitudinal electromagnetic waves can exist at a boundary or in an enclosed space.

The translation by Alexander J. Ellis was first published in 1875 (the first English edition was from the 1870 third German edition; Ellis's second English edition from the 1877 fourth German edition was published in 1885; the 1895 and 1912 third and fourth English editions were reprints of the second).

In 1849, while at Königsberg, Helmholtz measured the speed at which the signal is carried along a nerve fibre. At that time most people believed that nerve signals passed along nerves immeasurably fast. He used a recently dissected sciatic nerve of a frog and the calf muscle to which it attached. He used a galvanometer as a sensitive timing device, attaching a mirror to the needle to reflect a light beam across the room to a scale which gave much greater sensitivity. Helmholtz reported transmission speeds in the range of 24.6 - 38.4 meters per second.

Whoever, in the pursuit of science, seeks after immediate practical utility may rest assured that he seeks in vain. — Academic Discourse (Heidelberg 1862)