Meyerhof contracted frog muscles at low temperature and chemically quantified lactic acid concentration and oxygen consumption. He demonstrated a “fixed relation” of roughly three moles of lactic acid metabolized per mole of oxygen, proposing a quantitative model of aerobic recovery. The work suggested that lactic acid produced anaerobically is later oxidized in mitochondria, forming the basis of metabolic pathway charts. Predicting the oxygen needed for muscle recovery informs exercise load settings and rehabilitation plans. Meyerhof’s data became key evidence for the later-discovered Cori cycle and for validating individual glycolytic reactions. His methodology greatly contributed to standardizing chemical analysis techniques for metabolic products.

In his experiments, Otto Meyerhof repeatedly stimulated blood-depleted frog calf muscles immersed in Ringer solution and quantified lactic acid by pH titration of tissue extracts. At the same time he monitored oxygen partial pressure with an electrochemical sensor, obtaining the oxygen recovery curve after contraction. The observed rate of lactate disappearance was linearly correlated with oxygen uptake, revealing a constant mol ratio of 1 O2 to 3 lactate. He applied this ratio to energy balance calculations and theoretically inferred the intermediates linking glycogen breakdown to ATP resynthesis. Meyerhof further hypothesized that lactate released to the blood could be reconverted to glucose in the liver, anticipating the later Cori cycle concept. His achievements influenced the naming of the glycolytic pathway (the Embden–Meyerhof pathway) and helped systematize metabolic chemistry. The then innovative combination of histochemical assays and gas analysis became a prototype for clinical blood-gas measurement techniques. Subsequent research has shown that lactate is not merely a waste product but an energy carrier enabling metabolic flexibility during exercise. Thus Meyerhof’s idea of a “fixed relationship” remains central in modern sports science and clinical metabolism studies.

Meyerhof suppressed in situ glycolysis by working at low temperature while electrically stimulating isolated muscle, obtaining high S/N data on the lactate-oxygen correlation. Using a Warburg manometer he simultaneously measured O2 uptake and CO2 release, quantifying the respiratory quotient drop accompanying lactate oxidation. The constant of 1 mol O2 per 3 mol lactate suggests that complete oxidation of glycogen → 2 lactate → CO2 + H2O can reclaim about ΔG ≈ 677 kJ. His analysis pioneered indirect quantification of phosphorylated intermediates and foreshadowed the Meyerhof–Lohmann shuttle concept. Combined with Fick-based muscle blood-flow measurements, the fixed ratio was applied to analyse in vivo oxygen delivery-utilization efficiency. In kinetic modelling of glycolysis, this lactate-oxygen coefficient serves as a constraint parameter, greatly improving steady-state estimations. It is also cited as a basal constant when discussing HIF1α signalling and lactate fluxes under exercise-induced hypoxia. The frog model Meyerhof employed remains a gold standard for isolated muscle tests due to fibre-type uniformity and appropriate metabolic scale. Recent 13C tracer studies and high-resolution respirometry have reproduced the proportionality, underscoring Meyerhof’s foresight.