The aim of this study was to evaluate the use of pulse contour analysis calibrated with lithium dilution in a single device (LiDCOTM) for measurement of cardiac output (Q ̇) during exercise in healthy volunteers. We sought to; (a) compare pulse contour analysis (PulseCO) and lithium indicator dilution (LiDCO) for the measurement of Q ̇ during exercise, and (b) assess the requirement for recalibration of PulseCO with LiDCO during exercise. Ten trained males performed multi-stage cycling exercise at intensities below and above ventilatory threshold before constant load maximal exercise to exhaustion. Uncalibrated PulseCO Q ̇ (Q ̇raw) was compared to that calibrated with lithium dilution at baseline (Q ̇baseline), during submaximal exercise below (Q ̇low) and above (Q ̇high) ventilatory threshold, and at each exercise stage individually (Q ̇exercise). There was a significant difference between Q ̇baseline and all other calibration methods during exercise, but not at rest. No significant differences were observed between other methods. Closest agreement with Q ̇exercise was observed for Q ̇high (bias ± limits of agreement: 4.8 ± 30.0%). The difference between Q ̇exercise and both Q ̇low and Q ̇raw was characterized by low bias (4–7%) and wide limits of agreement (> ± 40%). Calibration of pulse contour analysis with lithium dilution prior to exercise leads to a systematic overestimation of exercising cardiac output. A single calibration performed during exercise above the ventilatory threshold provided acceptable limits of agreement with an approach incorporating multiple calibrations throughout exercise. Pulse contour analysis may be used for Q ̇ measurement during exercise providing the system is calibrated during exercise.