This randomized clinical trial aimed to evaluate the effect of two rotaries (ProTaper Universal Retreatment (PTUR)), D-Race (DR) + XP-Endo Finisher R (XPFR) and one reciprocating (Reciproc Blue (RB) retreatment techniques on the release of neuropeptides (Substance P, calcitonin gene-related peptide (CGRP)), and cytokines (IL-6 and IL-10) in periapical fluid in root canal retreatment of single-rooted teeth. In this randomized clinical trial (ClinicalTrials.gov ID: NCT05039502), seventy-five patients scheduled for retreatment were randomly divided into 3 groups according to the file system used to remove root canal filling materials (n = 25): PTUR, RB, and DR + XPFR. After reshaping and disinfection of the root canals, periapical fluid samples were taken, and the levels of Substance P, CGRP, IL-6, and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) test. Data were analyzed using the Kruskal-Wallis and chi-square tests. The level of significance was set as p = 0. 05. All the allocated participants received the intervention and were analyzed. There was no statistically significant difference among groups in terms of gender, age, tooth localization, and the distribution of analgesic use after treatment (p values 0.799, 0.095, 0.637, 1.000, respectively). No statistically significant difference was found in terms of the levels of Substance P, CGRP, and IL-10 among groups (p > .05), except IL-6. PTUR, RB, and DR + XPFR files have comparable results in the expression of inflammatory mediators. Retreatment files powered with rotary or reciprocating motion produced similar neuropeptide and cytokine levels in patients.