Presently there is limited data examining the relationship between physiological variables obtained during incremental exercise tests and cycle time trial performance over distances greater than 60-min. The purpose of this study was to determine the relationship between peak power output (Wpeak), the workload at the lactate threshold (WLT) and the average power output obtained during a 90-min (W90-min) cycle time trial (TT). Nine male welltrained cyclists (Age 32.1 ± 3.0 yrs) with a Wpeak of > 4 W/kg were recruited for the investigation. Each cyclist performed three tests over a two-week period. The tests included (1) a continuous incremental ramp test for determination of maximal oxygen uptake (VO2max; L/min); (2) a continuous incremental lactate test to measure W peak (W) and WLT (W); and (3) a 90-min TT to obtain the W90-min (W). The W90-min was significantly (p < 0.01) correlated with Wpeak (r = 0.91) and WLT (r = 0.91). However W90-min was not significantly related to VO2max (r = 0.38). In addition, Wpeak was highly related to WLT (r = 0.86; p < 0.01) but not significantly related to VO2max (r = 0.51; p = 0.16). The results of this study demonstrate that the maximum workload and the workload at the lactate threshold obtained during an incremental exercise test are highly related to 90-min TT performance in well-trained cyclists. The results also suggest that delaying blood lactate accumulation influences the maximum workload obtained during incremental cycle exercise. In contrast to other physiological variables, the WLT and Wpeak should be used to assess changes in cycling performance in highly trained athletes.