To determine the accuracy and precision of constant infusion transpulmonary thermodilution cardiac output (CITT-Q) assessment during exercise in humans, using indocyanine green (ICG) dilution and bolus transpulmonary thermodilution (BTD) as reference methods, cardiac output (Q) was determined at rest and during incremental one- and two-legged pedaling on a cycle ergometer, and combined arm cranking with leg pedaling to exhaustion in 15 healthy men. Continuous infusions of iced saline in the femoral vein (n= 41) or simultaneously in the femoral and axillary (n= 66) veins with determination of temperature in the femoral artery were used for CITT-Q assessment. CITT-Q was linearly related to ICG-Q (r= 0.82, CITT-Q = 0.876 × ICG-Q + 3.638,P< 0.001; limits of agreement ranging from −1.43 to 3.07 L/min) and BTD-Q (r= 0.91, CITT-Q = 0.822 × BTD + 4.481 L/min,P< 0.001; limits of agreement ranging from −1.01 to 2.63 L/min). Compared with ICG-Q and BTD-Q, CITT-Q overestimated cardiac output by 1.6 L/min (≈ 10% of the mean ICG and BTD-Q values,P< 0.05). For Q between 20 and 28 L/min, we estimated an overestimation < 5%. The coefficient of variation of 23 repeated CITT-Q measurements was 6.0% (CI: 6.1–11.1%). In conclusion, cardiac output can be precisely and accurately determined with constant infusion transpulmonary thermodilution in exercising humans.