To quantify the effect of two single-file reciprocating root canal preparation systems on Substance P (SP) and Calcitonin gene-related peptide (CGRP) expression in healthy human periodontal ligament (PDL).Forty PDL samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Prior to extraction, 20 of these premolars were divided equally in two groups, and then, root canals were prepared using one of two different single-file systems: WaveOne and Reciproc. Ten premolars were prepared with hand files and served as a positive control group. The remaining 10 premolars where extracted without treatment and served as a negative control group. All PDL samples were processed, and SP and CGRP were measured by radioimmunoassay.Greater SP and CGRP expression were found in the hand instrumentation group (1.220 pmol SP and 0.084 pmol CGRP per mg of PDL), followed by the WaveOne group (0.908 pmol SP and 0.046 pmol CGRP per mg of PDL) and the Reciproc group (0.511 pmol SP and 0.022 pmol CGRP per mg of PDL). The lower SP and CGRP values were associated with the intact control group (0.453 pmol SP and 0.018 pmol CGRP per mg of PDL). The Kruskal-Wallis test revealed significant differences between groups (P < 0.001). Post hoc Tukey HSD tests revealed significant differences in SP and CGRP expression between intact teeth in the control group and all the other groups (P < 0.001) except with the Reciproc group (P = 0.165 and P = 0.42 for SP and CGRP, respectively). Hand instrumentation was associated with significant differences with all the other groups (P < 0.001). Differences between the WaveOne and Reciproc groups were also significant (P < 0.001).Substance P and CGRP expression in PDL cells increased when teeth were prepared with WaveOne as well as with hand instrumentation. Reciproc maintained SP and CGRP levels in line with the negative control group.