Introduction <p>Tension-type headache (TTH) is the most prevalent primary headache disorder worldwide, yet its neurobiological underpinnings remain partially understood. Neurotransmitter and neuropeptide alterations have been proposed as contributing factors, but evidence remains inconsistent. This systematic review aims to synthesize the available evidence on peripheral and central neurotransmitter alterations in patients with TTH, and to identify potential neurochemical targets for future investigation.</p> Methods <p>We searched PubMed and Embase (Ovid) for studies reporting levels of neurotransmitters or neuropeptides in human samples from individuals with TTH. A total of 30 studies were included. Data on study design, sample type, and measured neuromodulators were extracted and narratively synthesized.</p> Results <p>No single neurotransmitter or neuropeptide emerged as a consistent biomarker or central mediator of TTH. However, some systems showed recurring alterations. Substance P levels were elevated in both salivary and platelet samples. Findings on endogenous opioids were mixed, with β-endorphins often reduced and methionine-enkephalin (MET) elevated, possibly reflecting compensatory responses. Serotonin data were heterogeneous and inconclusive, whereas nitric oxide may play a role in headache induction, independent of calcitonin gene-related peptide (CGRP).</p> Conclusions <p>Despite variability in results, substance P, endogenous opioids, and nitric oxide emerged as the most promising targets for further studies. Future research should prioritize standardized methodologies to clarify the role of these pathways in TTH pathophysiology.</p>