The normal circadian rhythm in exercise performance may be altered by the habitual timing of training. We have investigated if morning time trial performance is affected by the time at which moderate exercise is performed on the previous day. Eight male cyclists undertook two separate exercise sessions of sub-maximal cycle ergometry (60% V.O2peak for 30 min) at 07:00 h and 12:00 h the day before a 16.1-km time trial at 07:00 h. Heart rate, power output, ratings of perceived exertion, and rectal temperature were measured at rest and every 5 min in the pre-time trial exercises, and every 1.61 km during the time trial. Blood samples were taken at rest and immediately after the time trial for the measurement of lactate concentration. The time trial performed the day after the 07:00 h sub-maximal exercise was completed in 1672+/-135 s, compared to 1706+/-159 s for the time trial performed the day after the noon pre-time trial exercise (p=0.027). The time trial after exercise the previous morning was associated with higher work-rates (p=0.031), a higher net lactate accumulation after the time trial (p=0.018), and a trend for higher heart rates (p=0.093) compared to the time trial after exercise the previous noon. These findings suggest that cycling performance in an early morning time trial is improved if an athlete participates in early-morning rather than noontime moderate exercise the day before. This finding cannot be attributed to the physiological responses to the exercise on the pre-time trial day or to environmental factors. It is suggested that it might partly reflect an advantage gained by performing exercise in the day(s) immediately beforehand at the same time as the competition.