The efficiency of oxidative phosphorylation is determined by the P/O ratio, which is a measure of the molecules of ATP made per pair of electrons carried through the electron transport. Experimentally, researchers isolate mitochondria from cells, then use oxygen electrodes to determine the amount of ATP synthesized per oxygen molecule reduced to water molecules in the last step of the electron transport system.

The relative amounts of ATP made per pair of electrons entering the ETS varies. Electrons entering the system at complex I from NADH have a P/O ratio of about 3/1. Electrons entering the system at complex II (FAD's electrons) have a P/O ratio of about 2/1.

With this in mind, the balanced equation for the mitochondrial oxidation of NADH is as follows:

NADH + 4H⁺  + 1/2 O2 + 3ADP + 3Pi <=> NAD⁺ + 4H2O + 3 ATP ( = -220 kJ/mol)

Under standard conditions, the formation of ATP from ADP and Pi requires 31 kJ/mol. Therefore, coupling the synthesis of three ATPs to the oxidation of one NADH traps 93 kJ, or about 42%, of the energy released (under standard conditions).