<p>Various strategies have been employed to alleviate postoperative-endodontic pain (POP), with cryotherapy emerging as a promising approach. This randomized clinical trial aimed to evaluate the effectiveness of cold sodium hypochlorite (NaOCl) irrigation at 2.5&#xa0;°C throughout chemo-mechanical preparation in reducing POP intensity and substance P (SP) levels in patients with irreversible pulpitis and symptomatic apical periodontitis. Following administration of anesthesia and determining the working length, the first apical fluid sample was collected. The biomechanical preparation was carried out based on the group allocation. The Cold/Irr group received irrigation with cold 2.5% NaOCl at 2.5&#xa0;°C throughout the procedure and a final rinse with cold 2.5&#xa0;°C saline for 10&#xa0;min. The Cryo group received irrigation with 2.5% NaOCl at room temperature and a final rinse with cold 2.5&#xa0;°C saline for 10&#xa0;min. The control group received irrigation with 2.5% NaOCl at room temperature and final rinse with room temperature saline. Second sample of apical fluid was obtained prior to the obturation. Patients filled out a Visual Analog Scale (VAS) questionnaire to evaluate POP. The levels of SP were determined using the enzyme-linked immunosorbent assay (ELISA). The VAS scores in the control group were significantly higher than those in the other groups (<i>p</i> &lt; 0.001). Also, the control group exhibited significantly higher analgesics intake compared to other groups (<i>p</i> = 0.001). The Cold/Irr group demonstrated a significant decrease in the percentage of SP (<i>p</i> &lt; 0.001). A strong positive correlation was observed between SP and pain levels before and 6&#xa0;h after treatment. Cryotherapy techniques offer a straightforward approach to reducing POP and SP and alleviating the need for analgesics in patients with symptomatic apical periodontitis. </p><p>The trial registration number at ClinicalTrials.gov is NCT06106386 with initial release date 24/10/2023.</p>