This study aimed to investigate the acute effects of the Kinesio tape (KT) application on functional performance in healthy athletes. In this randomized, controlled crossover trial, a total of sixteen healthy soccer players (male = 14; age = 23.28 ± 3.13 years old) were assigned randomly into either KT over quadriceps, KT over hamstring, KT over quadriceps plus hamstring, or no intervention control condition. Four conditions were applied in a crossover design through three consecutive test sessions for each condition with a washout period of 2 days between the trials. Afterwards, all participants performed a 5-min warm-up routine and four sets of 30-s static stretching exercises for the hamstring and gluteal muscles for three consecutive treatment sessions. The running, jump, and flexibility tests were used to assess the functional performance of healthy athletes. A total of sixteen participants completed the study. No significant differences in the jump, flexibility, and running tests among the conditions were reported (p > 0.05). These findings suggest that KT application has no acute effects in improving functional performance in healthy athletes. However, further studies with larger sample sizes are needed to verify our results.