Introduction. Diacutaneous Fibrolysis is defined as specific instrumental intervention to normalize function in the musculoskeletal system. It is considered a treatment method for the mechanical alterations of the locomotor system, and it is widely used in sports for therapeutic and preventive purposes. Despite the clinical benefits observed in different musculoskeletal conditions, the action mechanism of diacutaneous fibrolysis remains uncertain. There are no studies evaluating the neuromuscular response on the posterior muscular chain of the lower extremity in athletes, where overload, stiffness, and injury incidence are high. Objective. To evaluate the immediate, and 30 min post treatment effects of a single diacutaneous fibrolysis session on passive neuromuscular response and mechanosensitibity on hamstring and gluteus in athletes with shortening. Design. A randomized within participant clinical trial. Methods. Sixty-six athletes with hamstring shortening were included (PKE < 160). The lower limbs were randomized between the experimental limb and control limb, regardless of dominance. A single session of diacutaneous fibrolysis was applied to the posterior gluteus maximus, biceps femoris, and semitendinosus of the experimental lower limb whereas the control limb was not treated. Viscoelastic muscle properties (myotonometry), contractile muscle properties (tensomiography), and mechanosensitivity (algometry) were tested before treatment (T0), after treatment (T1), and 30 min post treatment (T2). Results. Regarding viscoelastic properties, in the intra-group analysis we found statistically significant differences in the experimental limb at T1, decreasing muscle stiffness in gluteus maximus (p < 0.042), in biceps femoris (p < 0.001) and in semitendinosus (p < 0.032). We also observed statistically significant differences in Tone decrease (p < 0.011) and relaxation increase (p < 0.001) in biceps femoris. At T2, the decrease in stiffness in all tested muscles was maintained (p < 0.05). There were statistically significant inter-groups differences in stiffness on gluteus (p < 0.048) and biceps femoris (p < 0.019) and in tone on biceps femoris (p < 0.009) compared to the control limb. For contractile properties, we only found statistically significant differences on maximal radial displacement (Dm) in gluteus, both control and experimental at T2 (p < 0.05) and in biceps femoris control (p < 0.030). No changes were found in the mechanosensitivity. Conclusions. A single session of diacutaneous fibrolysis produces changes in some parameters related to viscoelasticity properties of the biceps femoris and gluteus. There were no changes on contractile properties on semitendinosus. Only small changes on the contractile properties on the gluteus maximus and biceps femoris were found. No effect was found on the mechanosensitivity of the posterior chain muscles in athletes with hamstring shortening.