Background Accurate assessment of cardiac output is critical to the diagnosis and management of various cardiac disease states; however, clinical standards of direct Fick and thermodilution are invasive. Noninvasive alternatives, such as closed-circuit acetylene (CH) rebreathing, warrant validation. Methods and Results We analyzed 10 clinical studies and all available cardiopulmonary stress tests performed in our laboratory that included a rebreathing method and direct Fick or thermodilution. Studies included healthy individuals and patients with clinical disease. Simultaneous cardiac output measurements were obtained under normovolemic, hypovolemic, and hypervolemic conditions, along with submaximal and maximal exercise. A total of 3198 measurements in 519 patients were analyzed (mean age, 59 years; 48% women). The CH method was more precise than thermodilution in healthy individuals with half the typical error (TE; 0.34 L/min [=0.92] and coefficient of variation, 7.2%) versus thermodilution (TE=0.67 [=0.70] and coefficient of variation, 13.2%). In healthy individuals during supine rest and upright exercise, CH correlated well with thermodilution (supine: =0.84, TE=1.02; exercise: =0.82, TE=2.36). In patients with clinical disease during supine rest, CH correlated with thermodilution (=0.85, TE=1.43). CH was similar to thermodilution and nitrous oxide (NO) rebreathing technique compared with Fick in healthy adults (CH rest: =0.85, TE=0.84; CH exercise: =0.87, TE=2.39; thermodilution rest: =0.72, TE=1.11; thermodilution exercise: =0.73, TE=2.87; NO rest: =0.82, TE=0.94; NO exercise: =0.84, TE=2.18). The accuracy of the CH and NO methods was excellent (=0.99, TE=0.58). Conclusions The CH rebreathing method is more precise than, and as accurate as, the thermodilution method in a variety of patients, with accuracy similar to an NO rebreathing method approved by the US Food and Drug Administration.