The relationship of oxygen uptake [Formula: see text] to ventilation [Formula: see text], i.e., oxygen uptake efficiency (OUE) is known to differ between normal subjects and patients with congestive heart failure. However, only the oxygen uptake efficiency slope (OUES, i.e., slope of [Formula: see text] has previously been reported. To understand the physiology and to improve the usefulness of OUE in assessing cardiovascular function, we analyzed the complete response pattern of OUE during entire incremental exercise tests and ascertained effect of age, body size, gender, fitness, and ergometer type on exercise OUE to generate reference values in normal healthy subjects. We investigated the effect of age, gender, and fitness on OUE using incremental cardiopulmonary exercise in 474 healthy subjects, age 17-78 years, of which 57 were highly fit. The final methods of OUE analysis were: (1) OUE plateau at the highest values (OUEP), (2) OUE at anaerobic threshold (OUE@AT), and (3) OUES using the entire exercise period. The OUEP and OUE@AT were similar, highly reproducible, less variable than the OUES (p < 0.0001), and unaffected by the study sites or types of ergometry. The resultant prediction equations from 417 normal subjects for men were OUEP (mL/L) = 42.18 - 0.189 × years + 0.036 × cm and OUES [L/min/log(L/min)] = -0.610 - 0.032 × years + 0.023 × cm + 0.008 × kg. For women, OUEP (mL/L) = 39.16 - 0.189 × years + 0.036 × cm and OUES [L/min/log(L/min)] = -1.178 - 0.032 × years + 0.023 × cm + 0.008 × kg. OUE@AT was similar to OUEP. Extreme fitness has a minimal effect on OUEP. OUEP is advantageous, since it measures maximal oxygen extraction from ventilated air but does not require high intensity exercise. The OUEP is a non-invasive parameter dependent only on age, gender, height, and cardiovascular health.