OBJECTIVE The purpose of this study was to determine the effect of targeted eccentric calf muscle exercises compared to regular training on ankle dorsiflexion in healthy adolescent soccer players with a decreased ankle dorsiflexion. METHODS Male adolescent players (aged 14-21 years) from two professional soccer clubs were evaluated with the Weight Bearing Dorsiflexion Lunge Test (WBDLT) at baseline and after 12 weeks of this prospective controlled study. One club served as the control group and the other as the intervention group. Players with decreased ankle dorsiflexion (WBDLT) ≤ 10 cm) performed stretching and eccentric calf muscle exercises three times per week next to regular training in the intervention group, and performed only regular training in the control group. Primary outcome was the between-group difference in change in WBDLT between baseline and 12 weeks. RESULTS Of 107 eligible players, 47(44 %) had a decreased ankle dorsiflexion. The WBDLT (± standard deviation) increased in the intervention group from 7.1 (± 1.8) to 7.4 (± 2.4) cm (95 % Confidence Interval (CI)[-0.493 to 1.108], p = 0.381) and in the control group from 6.1 (± 2.4) to 8.2 (± 2.9) cm (95 % CI [1.313 to 2.659], p < 0.001). The difference in change of WBDLT between both groups was statistically significant (95 % CI [-2.742 to -0.510], p = 0.005). CONCLUSIONS Targeted eccentric calf muscle exercises do not increase ankle dorsiflexion in healthy adolescent soccer players. Compared to regular training, eccentric exercises even resulted in a decreased calf muscle flexibility. BACKGROUND This trial was registered retrospectively on the 7th of September 2016 in The Netherlands Trial Register (ID number: 6044).