Background: Stretching techniques for hamstring muscles have been described both to increase muscle length and to evaluate nerve mechanosensitivity. Aim: We sought to evaluate the short-term effects of three types of hamstring stretching on hamstring length and report the type of response (neural or muscular) produced by ankle dorsiflexion and perceived sense of effort in asymptomatic subjects. Methods: A randomised cross-over clinical trial was conducted. A total of 35 subjects were recruited (15 women, 20 men; mean age 24.60 ± 6.49 years). Straight leg raises (SLR), passive knee extensions (PKE), and maximal hip flexion (MHF) were performed on dominant and non-dominant limbs. In addition, the intensity of the applied force, the type and location of the response to structural differentiation, and the perceived sensation of effort were assessed. Results: All stretching techniques increased hamstring length with no differences between limbs in the time*stretch interaction (p < 0.05). The perceived sensation of effort was similar between all types of stretching except MHF between limbs (p = 0.047). The type of response was mostly musculoskeletal for MHF and the area of more neural response was the posterior knee with SLR stretch. Conclusions: All stretching techniques increased hamstring length. The highest percentage of neural responses was observed in the SLR stretching, which produced a greater increase in overall flexibility.