To improve flexibility, stretching is most commonly used and in training interventions duration-dependent effects are hypothesized. However, there are strong limitations in used stretching protocols in most studies, particularly regarding documentation of intensity and performed procedure. Thus, aim of this study was to compare different stretching durations on flexibility in the plantar flexors and to exclude potential biases. Eighty subjects were divided into four groups performing daily stretching training of 10min (IG10), 30min (IG30) and 1h (IG60) and one control group (CG). Flexibility was measured in bended and extended knee joint. Stretching was performed with a calf muscle stretching orthosis to ensure long-lasting stretching training. Data were analysed with a two-way ANOVA for repeated measures on two variables. Two-way ANOVA showed significant effects for time (ƞ² = 0.557-0.72, p < 0.001) and significant interaction effects for time x group (ƞ² = 0.39-0.47, p < 0.001). Flexibility in the knee to wall stretch improved with 9.89-14.46% d = 0.97-1.49 and 6.07-16.39% with d = 0.38-1.27 when measured via the goniometer of the orthosis. All stretching times led to significant increases in flexibility in both tests. While there were no significant differences measured via the knee to wall stretch between the groups, the range of motion measurement via the goniometer of the orthosis showed significantly higher improvements in flexibility depending on stretching duration with the highest increase in both tests with 60 minutes of stretch per day.