Pacing during a high-intensity cycling time trial (TT) appears to prevent premature task failure, but the performance fatigability during a self-paced exercise is currently unknown. Therefore, the current study characterized the time course of performance fatigability during a 4-km TT. Eleven male cyclists performed three separated TTs in a crossover, counterbalanced design. The TTs lasted until the end of the fast-start (FS; 600 ± 205 m), even-pace (EP; 3,600 ± 190 m), and end-spurt (ES; 4,000 m) phases. Performance fatigability was characterized by using isometric maximal voluntary contractions (IMVCs), whereas the muscle activation [i.e., voluntary activation (VA)] and contractile function of knee extensors [e.g., peak torque of potentiated twitches (TwPt)] were evaluated using electrically evoked contractions performed before and 1 min after each specific part of the trial. Gas exchange, power output (PO), and electromyographic activity (EMG) were also recorded. EMG/PO showed an abrupt increase followed by a continuous decrease toward the end of FS, resulting in a drop in IMVC (−12%), VA (−8%), and TwPt (−23%). EMG/PO was stable during EP, with no additional drop on IMVC, VA, or TwPt (−12%, −6%, and −22%, respectively). EMG/PO increased abruptly during the ES, but there was no change in IMVCs, VA, or TwPt (−13%, −8%, and −26%, respectively). These findings demonstrate that the performance fatigability during a self-paced exercise is characterized by a large drop in contractile function and muscle activation at the beginning of the trial (i.e., FS), without additional change during the middle and end phases (i.e., EP and ES). NEW & NOTEWORTHY The time course of performance fatigability throughout a self-paced exercise is currently unknown. The results showed that a large amount of muscle activation and contractile function impairments are attained early on a self-paced exercise (first ∼15% of the total time trial distance) and maintained throughout the test. This novel finding characterizes the performance fatigability from a contractile function and muscle activation perspective, which brings new insights for future studies focused on real-world exercise training and competition.