Working at the University of Berlin (1836–96) under Johannes Müller, whom he later succeeded as professor of physiology (1858), Du Bois-Reymond studied fishes that are capable of generating electrical currents. Turning to the study of electrical conduction along nerve and muscle fibres, he found (1843) that a stimulus applied to the electropositive surface of the nerve membrane causes a decrease in electrical potential at that point and that this “point of reduced potential”—the impulse—travels along the nerve as a “wave of relative negativity.” He immediately was able to demonstrate that this phenomenon of “negative variation” also occurs in striated muscle and is the primary cause of muscular contraction. Although later research showed the process of nerve and muscle stimulation to be much more complex than Du Bois-Reymond's model, the summation of his studies in Untersuchungen über thierische Elektricität, 2 vol. (1848–1884; “Researches on Animal Electricity”), created the field of scientific electrophysiology.

Du Bois-Reymond's intellectual collaboration with Hermann von Helmholtz, Carl Ludwig, and Ernst von Brücke proved to be of great importance to the course of German physiology and to biological thought in general. At the university, their biophysics program, designed to reduce physiology to applied physics and chemistry, influenced the psychological theories of Sigmund Freud and did much to purge physiology of vitalistic theories that depicted all organic matter as arising from a “life force” peculiar to living things and quite different from all known physical phenomena.