Background: Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with a pressing shortage of therapies. Exercise intolerance is a cardinal symptom of HFpEF, yet its pathophysiology remains uncertain. Methods: We investigated the mechanism of exercise intolerance in 134 patients referred for cardiopulmonary exercise testing: 79 with HFpEF and 55 controls. We performed cardiopulmonary exercise testing with invasive monitoring to measure hemodynamics, blood gases, and gas exchange during exercise. We used these measurements to quantify 6 steps of oxygen transport and utilization (the O 2 pathway) in each patient with HFpEF, identifying the defective steps that impair each one's exercise capacity (peak Vo 2 ). We then quantified the functional significance of each O 2 pathway defect by calculating the improvement in exercise capacity a patient could expect from correcting the defect. Results: Peak Vo 2 was reduced by 34±2% (mean±SEM, P<0.001) in HFpEF compared with controls of similar age, sex, and body mass index. The vast majority (97%) of patients with HFpEF harbored defects at multiple steps of the O 2 pathway, the identity and magnitude of which varied widely. Two of these steps, cardiac output and skeletal muscle O 2 diffusion, were impaired relative to controls by an average of 27±3% and 36±2%, respectively (P<0.001 for both). Due to interactions between a given patient's defects, the predicted benefit of correcting any single one was often minor; on average, correcting a patient's cardiac output led to a 7±0.5% predicted improvement in exercise intolerance, whereas correcting a patient's muscle diffusion capacity led to a 27±1% improvement. At the individual level, the impact of any given O 2 pathway defect on a patient's exercise capacity was strongly influenced by comorbid defects. Conclusions: Systematic analysis of the O 2 pathway in HFpEF showed that exercise capacity was undermined by multiple defects, including reductions in cardiac output and skeletal muscle diffusion capacity. An important source of disease heterogeneity stemmed from variation in each patient's personal profile of defects. Personalized O 2 pathway analysis could identify patients most likely to benefit from treating a specific defect; however, the system properties of O 2 transport favor treating multiple defects at once, as with exercise training.