This systematic review and meta-analysis investigated the influence of the number of files (full-sequence rotary-file versus reciprocating single-file systems) used during root canal preparation on the apical extrusion of debris and its biological relationship with the occurrence of symptomatic apical periodontitis. An extensive literature research was carried out in the Medline, ISI Web of Science and Cochrane databases, for relevant articles with the keyword search strategy. Based on inclusion and exclusion criteria, two reviewers independently rated the quality of each study determining the level of evidence of the articles selected. The primary outcome for the meta-analysis was determined by the amount of debris extruded into the periapical tissue during root canal preparation with multiple- or single-file systems in four laboratory studies. Analysis of in vivo release of neuropeptides (SP and CGRP) after root canal preparation with single- or multiple-file systems was also carried out. Amongst the 128 articles initially found, 113 were excluded for being nonrelevant or not fulfilling the selection criteria. Another four articles were excluded after methodology evaluation. Finally, nine laboratory studies and two in vivo studies were included in the systematic review. Four of the laboratory studies were further included for meta-analysis that revealed greater debris extrusion after the use of single-file techniques when compared to multiple-file systems. Analysis of in vivo neuropeptide expression in the periodontal ligament suggests that the design of the instrument is more important than the number of files used. Both rotary and reciprocating single-file systems generate apical extrusion of debris in laboratory studies, or expression of neuropeptides in vivo. Available evidence is limited, but supports the fact that this inflammatory reaction is not influenced by the number of files but the type of movement and the instrument design.