This study examined whether circadian rhythms affect lactate threshold (Thlac) during rowing exercise. Eleven male, endurance-trained athletes [mean (SD) age 29.5 (6.1) years] rowed at 0200, 0600, 1000, 1400, 1800 and 2200 hours under the same experimental conditions. Capillary blood (25 μl) was obtained from the tip of the toe during the last 30 s of a continuous, multi-stage, 3-min, incremental protocol on the Concept II ergometer. To determine Thlac, a curve-fitting procedure (the Dmax method), a visual method (Thlac-vis) and the fixed blood lactate concentration of 4.0 mmol l−1 (Thlac-4 mM) were used. Circadian rhythms were apparent for oxygen consumption and heart rate at Thlac using the Dmax method (P=0.02 and P=0.04 respectively), with the acrophases at 2139 hours and 2032 hours respectively coinciding in phase with that of core body temperature. The conclusion is that tests should be completed at the same time of day at which the athlete usually trains, to ensure precision of Thlac determination, especially when the Dmax method is used to determine Thlac.