Purpose: To investigate how cycling time-trial (TT) performance changes over a cycling season, both in a “fresh” state and in a “fatigued” state (durability). Additionally, the aim was to explore whether these changes are related to changes in underlying physiological factors such as gross efficiency, energy expenditure (EE), and substrate oxidation (fat oxidation [FatOx] and carbohydrate oxidation [CarbOx]). Methods: Sixteen male semiprofessional cyclists visited the laboratory on 3 occasions during a cycling season (PRE, START, and IN) and underwent a performance test in both fresh and fatigued states (after 38.1 [4.9] kJ/kg), containing a submaximal warm-up for the measurement of gross efficiency, EE, FatOx, and CarbOx and a maximal TT of 1 (TT1min) and 10 minutes (TT10min). Results were compared across states (fresh vs fatigued) and periods (PRE, START, and IN). Results: The average power output (PO) in TT1min decreased (P < .05) from fresh to fatigued state across all observed periods, whereas there was no change in the PO in TT10min. Over the course of the season, the PO in TT1min in the fatigued state improved more compared with the PO in TT1min in the fresh state. Furthermore, while EE did not significantly change, there was an increase in FatOx and a decrease in CarbOx toward the fatigued state. These changes diminished during the cycling season (IN), indicating a greater contribution of CarbOx in the fatigued state. Conclusions: TT1min performance is more sensitive to fatigue compared with TT10min. Also, during a cycling season, durability improves more when compared with fresh maximal POs, which is also observed in the changes in substrate oxidation.