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Background: Myofascial trigger points (MTrPs) are hypersensitive nodules in a taut band (TB) of skeletal muscle. Dry needling (DN) is an invasive technique recommended for the treatment of MTrPs. However, to our knowledge, no studies have investigated the influence of the DN technique on modification of muscle stiffness and neurophysiological properties of MTrPs. Objective: The objective was to examine the effect of DN on muscle stiffness and motoneuron excitability of a latent medial MTrP (nodule and TB) of the soleus muscle in non-injured subjects. Methods: A double-blinded randomised controlled trial of 46 subjects with latent medial MTrPs of the soleus was conducted, in which all received one session of DN. The intervention group (n = 23) were subjected to DN into the MTrP (the nodule), while the control group (n = 23) were subjected to DN into the TB. Assessment was carried out at baseline (pre-test), after the intervention (post-test) and 1 week after the intervention (follow-up). Biomechanical variables (muscle resistive force at 10°/s and 180°/s, muscle extensibility and strength), as measured with an isokinetic dynamometer, and neurophysiological variables (H-reflex), were recorded. Results: There were no statistically significant differences in biomechanical or neurophysiological assessments between groups. Considering the intra-group analysis, subjects in the intervention group exhibited increased maximal isometric voluntary force to ankle plantarflexion (MIVFp) at both post-intervention and follow-up assessment (p Conclusion: One session of DN targeting latent MTrPs did not change muscle stiffness, muscle extensibility or motoneuron excitability. Further research on subjects with muscle tone disorders should be considered to better address the impact of DN on muscle tone. Trial registration number: NCT02575586 (ClinicalTrials.gov).