Resumo. ABSTRACT This study aimed to analyze the anaerobic performance and muscle activation during a supramaximal cycling test at three different saddle positions. Twelve competitive cyclists completed an incremental cycling test and three 30-s Wingate tests in three different saddle heights (reference, downward, and upward), in a random order, on different days. The saddle height was individually shifted downward and upward (seeing 2.5% of the distance from the pubic symphysis to ground) from the reference position. The electromyographic signal (EMG) data was obtained from the rectus femoris, vastus lateralis, biceps femoris (long head), and gastrocnemius lateralis in order to assess muscle activation during the entire test. The anaerobic variables and the EMG data were divided into six consecutive windows of 5-s. The EMG signals were normalized by the first 5-s window of the reference position to provide the percentage changes throughout the test. The results suggest that during a 30-s Wingate test small changes in saddle height result in greater peak power output (reference=1380±241 W; downward=1497±175 W, p=0.036; upward=1491±225 W, p=0.049) and greater activation period for vastus lateralis (reference=33.6%, downward=33.2%, upward=35.0%; p=0.001) in comparision to rectus femoris (reference=24.5%, downward=25.2%, upward=23.7%), biceps femoris (reference=20.7%, downward=20.8%, upward=19.9%), and gastrocnemius lateralis (reference=21.2%, downward=20.8%, upward=19.9%). The results suggest that small adjustments in saddle height may affect the force-length relationship of the muscles of the lower limb, and consequently their recruitment pattern and their ability to generate force.