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. 2025 Jun 30;14(4):1279–1291. doi: 10.1007/s40122-025-00761-3

Neurotransmitter Imbalance in Tension-Type Headache: A Systematic Review of Mechanisms and Therapeutic Targets

Lanfranco Pellesi 1,✉, Aidin Yangjeh 1, Ibrahim Hajjaj 1, Mousbah Lababidi 1, Fezan Sarwar 1, Wei Wang 2, Paolo Martelletti 3
PMCID: PMC12279646  PMID: 40588693

Abstract

Introduction

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.

Methods

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.

Results

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).

Conclusions

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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40122-025-00761-3.

Keywords: β-Endorphins, Biomarker, CGRP, Nitric oxide, Neuropeptides, Serotonin, Substance P

Key Summary Points

Substance P is the most consistently altered neuropeptide in patients with tension-type headache (TTH).
The endogenous opioid system appears dysfunctional, possibly reflecting a compensatory response to impaired descending pain modulation.
Nitric oxide may contribute to headache induction as shown in provocation studies with glyceryl trinitrate.
Findings on serotonin levels are heterogeneous and inconclusive, with no consistent pattern across studies.
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Introduction

Tension-type headache (TTH) is the most common primary headache disorder globally and a leading cause of disability [1, 2]. Despite its high prevalence and clinical burden, the pathophysiological mechanisms underlying TTH remain only partially elucidated. While muscular and psychosocial factors have historically been implicated, accumulating evidence highlights the role of neurochemical imbalances, particularly involving neurotransmitters and neuropeptides that modulate pain and central sensitization [3]. Among the neurochemical systems investigated, alterations in serotonin (5-HT), gamma-aminobutyric acid (GABA), substance P, calcitonin gene-related peptide (CGRP), endogenous opioids, and nitric oxide (NO) have all been associated with nociceptive processing and pain thresholds in TTH. However, findings across studies have often been inconsistent. Understanding the involvement of these neurotransmitters and neuropeptides may provide insight into the mechanisms contributing to the chronification of TTH and identify potential biomarkers or therapeutic targets. This systematic review aims to synthesize the available evidence on neurotransmitter and neuropeptide alterations in patients with TTH. We examine studies reporting on levels of neuromodulators in human samples, across both interictal periods and active headache states. By organizing the evidence according to neurotransmitter systems, we seek to clarify the extent and consistency of biochemical alterations in TTH and to explore their relevance for future diagnostic and treatment strategies.

Methods

We conducted a systematic search of PubMed and Embase (via Ovid) on 14 February 2025. We used a combination of terms related to tension-type headache (e.g., “tension-type headache”, “muscle contraction headache”, “TTH”) and neurotransmitters (e.g., “substance P”, “serotonin”, “neuropeptides”, “CGRP”). The full search strategies are available in Table S1. A preliminary scoping of other databases such as Web of Science, Scopus and Cochrane Central did not reveal significant additional coverage relevant to our focused research question. Titles and abstracts of retrieved records were screened independently by at least two authors to identify studies meeting eligibility criteria and to exclude clearly irrelevant records. Decisions regarding exclusion were made using a majority voting system. Studies were selected for full-text review if they met the following criteria: (1) evaluation of neurotransmitter levels in human biological samples, (2) inclusion of a study population with TTH, and (3) availability of the full text in English. We excluded reviews, letters to the editor, conference abstracts, and oral communications. Reference lists of relevant narrative and systematic reviews were screened to identify any additional eligible studies not captured in the initial search. We also considered articles cited in the included studies and other literature known to be relevant by the authors. Following full-text assessment, studies were included in the final review if they reported measurement of at least one neurotransmitter in human samples collected from individuals with TTH. We included studies involving individuals of any age group (children, adolescents, adults) and both episodic and chronic forms of TTH, provided that neurotransmitter measurements were reported. When such distinctions were available, they were noted and considered in the synthesis. Owing to the heterogeneity in study designs, sample types, analytical techniques, and outcome measures, no formal risk of bias assessment was performed. Methodological limitations of the included studies were instead discussed narratively in the “Discussion” section. This review was not registered in PROSPERO, as it involved a qualitative synthesis of mechanistic studies and did not focus on clinical interventions. This study is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. For this reason, ethical approval is not required.

Data Extraction

Data extraction was performed independently by at least two authors, who reviewed the full texts to collect information on study design, sample characteristics, neurotransmitters assessed, and their levels. Discrepancies were resolved through collegial discussion until consensus was reached. We did not identify any overlapping or duplicate study populations among the included articles. The data extraction template is provided in Supplementary Table 2. Reference management, including deduplication, was performed using EndNote (version 21). The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [4].

Data Reporting

As this systematic review did not involve a meta-analysis or pooled statistical comparisons, no statistical analyses were performed. Data are presented as originally reported in the included studies. Quantitative data regarding neuropeptide levels are presented as reported in the source articles, as either means with standard deviations (mean ± SD), means with standard error of the mean (mean ± SEM), means with ranges, medians with quartiles, or medians with 95% confidence limits. Units of measurement, time of sample collection (e.g., during headache attacks or interictal periods), and biological sample types (e.g., plasma, saliva, or platelets) are preserved as described in the primary sources. Owing to heterogeneity in study designs and outcome measures, a narrative synthesis was performed to describe and contextualize findings across studies.

Results

Our search strategy yielded a total of 1244 records, of which 30 studies met the inclusion criteria and were included in the final review. After removing 52 duplicate records, we excluded 263 records that were identified as reviews, systematic reviews, meta-analyses, study protocols, guidelines, book chapters, conference proceedings, or oral presentations. An additional 789 records were excluded because they were not related to TTH, while 106 studies focusing on TTH were excluded because they did not assess neurotransmitters. We also excluded three preclinical studies conducted in animals. Following full-text assessment, one study was excluded because it included only participants with migraine, without a TTH subgroup [5]. In total, 30 studies were included in the qualitative synthesis (Fig. 1). Tables 1 and 2 summarize neurotransmitters found at increased and decreased levels, respectively, in patients with TTH compared with healthy controls. Figure 2 provides a graphical representation of these findings.

Fig. 1.

Fig. 1

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Flowchart of the systematic search

Table 1.

Neurotransmitters reported at higher levels in individuals with tension-type headache compared with healthy controls

Higher levels during headache Higher levels during headache-free periods
Substance P Platelets [8] and saliva [9] Platelets [7]
Metenkefalin Plasma [20] and cerebrospinal fluid [12, 21]
Serotonin Saliva [9] Platelets [26]
Nitric oxide Plasma [29]
Neuropeptide Y Plasma [34]
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Table 2.

Neurotransmitters reported at lower levels in individuals with tension-type headache compared with healthy controls

Lower levels during headache Lower levels during headache-free periods
β-Endorphin Peripheral blood mononuclear cells [7, 22] and plasma [17, 18, 22]
Metenkefalin Platelets [20]
Dynorphin Cerebrospinal fluid [12]
Serotonin Platelets [8] Platelets [7, 23, 24] and plasma [27]
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Fig. 2.

Fig. 2

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Neurotransmitter alterations in tension-type headache compared with healthy controls

Sensory Neuropeptides

Substance P, a sensory neuropeptide involved in trigeminal pain signaling, plays a role in headache pathophysiology. Plasma levels of substance P in the external jugular vein showed no significant differences between 20 patients with chronic TTH and 20 healthy controls, regardless of whether patients were experiencing a headache or not (median and quartiles: 1.5 pmol/L (0.3–1.7) versus 1.5 pmol/L (0.0–2.2) during headache; 1.7 pmol/L (1.1–1.9) versus 1.5 pmol/L (0.0–2.2) during interictal periods) [6]. A study examining platelet levels of substance P in 30 patients with TTH during interictal periods found significantly higher concentrations compared with 30 healthy controls (mean ± SD: 438.6 ± 72.7 pg/109 platelets versus 308.0 ± 42.9 pg/109 platelets) [7]. Additionally, platelet substance P levels showed a negative correlation with pain pressure thresholds measured on the temporal muscles of individuals with TTH [7]. During headache, the concentration of substance P in platelets was significantly higher in 31 patients with TTH than 27 healthy controls (mean ± SD: 305.2 ± 208.6 pg/109 platelets versus 148.6 ± 150.1 pg/109 platelets) [8]. In a separate study, salivary substance P levels were significantly elevated in 48 patients during active headache periods compared with 43 healthy controls (mean ± SD: 243.5 ± 1137 pg/mL versus 21.2 ± 17.4 pg/mL) [9]. CGRP is another sensory peptide involved in trigeminal pain signaling. Interictal plasma levels of CGRP measured in the peripheral circulation tended to be higher in 30 patients with chronic TTH than 34 control subjects, without reaching statistical significance (mean ± SEM: 63 ± 5 pmol/L versus 53 6 3 pmol/L, p = 0.06) [10]. A further study found that plasma levels of CGRP were not different between 50 patients with TTH and 50 healthy controls (p = 0.49) [11]. Similarly, plasma CGRP concentration was not different among patients with episodic and chronic TTH (p = 0.97) [11]. Cerebrospinal fluid (CSF) concentrations of CGRP did not differ between 41 patients with chronic TTH and 7 healthy controls (mean ± SD: 11.1 ± 3 pmol/L versus 11.8 ± 7 pmol/L) [12]. In a pediatric clinic, serum samples of CGRP did not differ between 59 children with TTH and 53 healthy controls (median and quartiles: 17.3 ng/mL (9.8–60.8) versus 20.4 ng/mL (12.9–63.9) during no headache) [13]. Somatostatin is another peptide involved in modulating nociceptive transmission and can reduce pain sensitivity within sensory pathways [14]. In a cross-sectional evaluation, CSF concentrations of somatostatin did not differ between 38 patients with chronic TTH and 8 healthy controls (mean ± SD: 23.9 ± 11.4 pmol/L versus 24.1 ± 13.8 pmol/L) [12].

Endogenous Opioids

Endogenous opioids, including β-endorphins and enkephalins, play a role in pain modulation and analgesia. In a study investigating their involvement in TTH, significantly lower β-endorphin levels were found in peripheral blood mononuclear cells (PBMCs, including lymphocytes and monocytes) of 30 patients during interictal periods compared with 30 healthy controls (mean ± SD: 28.2 ± 5.8 pg/106 cells versus 36.8 ± 4.9 pg/106 cells) [7]. Furthermore, β-endorphin levels showed a significant positive correlation with pressure pain thresholds measured on the temporal muscles of individuals with TTH [7]. In another study, levels of β-endorphins in PBMCs were not different between 23 patients with episodic TTH and 37 healthy controls (mean ± SEM: 34.27 ± 5.9 pg/106 PBMNCs versus 38.86 ± 3.42 pg/106 PBMNCs) [15]. Peripheral blood samples were taken from patients not enduring headache attacks and not undergoing prophylactic treatment [15]. Moreover, CSF and plasma concentration of β-endorphins were not different between 50 patients with chronic TTH and 44 age-matched controls (median and 95% confidence limits: 12.8 pmol/l (11.0–14.5) versus 11.9 pmol/L (10.9–14.2) and 3.1 (2.4–3.7) and 3.3 pmol/L (1.8–4.0) in CSF and plasma, respectively) [16]. Reduced plasma levels of β-endorphins were observed in two independent cohorts of patients with chronic TTH (8 patients compared with 12 healthy controls, mean ± SEM: 5.8 ± 0.9 pmol/L versus12.6 ± 1.3 pmol/L; 11 patients compared with 8 controls, mean ± SD: 16.8 ± 2.5 pg/mL versus 26.0 ± 6.1 pg/mL) [17, 18]. However, these findings were not replicated in a separate cohort of 11 patients, whose mean plasma β-endorphin levels remained within the normal range (mean ± SD: 18.1 ± 10.2 pg/mL) [19]. Outside of headache attacks, nine patients with TTH exhibited low platelet levels of metenkefalin (MET) and significantly elevated plasma MET levels compared with 9 healthy controls (7.6 × 10–21 g/platelet versus 12.8 × 10–21 g/platelet and 68.1 ng/L versus 39.6 ng/L in platelets and plasma, respectively) [20]. The median CSF level of MET immunoreactivity was significantly higher in 47 patients with chronic TTH than 47 headache-free control subjects (median and quartiles: 115 pmol/L (107–134) versus 79 pmol/L (73–87) [21]. No correlation was found between MET immunoreactivity and either pericranial tenderness, nociceptive flexion-reflex threshold, or thermal pain threshold [21]. Comparison between CSF concentrations from patients with chronic TTH and healthy subjects revealed significantly increased MET (47 patients versus 48 controls, mean ± SD: 119 ± 23 pmol/L versus 82 ± 19 pmol/L), and reduced dynorphin (38 patients versus 10 controls, mean ± SD: 30 ± 11 pmol/L versus 39 ± 8 pmol/L) in patients, whereas the concentrations of β-endorphin were similar (47 patients versus 44 controls, mean ± SD: 13.9 ± 5.0 pmol/L versus 12.2 ± 3.1 pmol/L) [12]. In a study conducted in a pediatric department, significantly lower plasma and PBMC levels of β-endorphins were observed in 7 children with episodic TTH compared with 17 headache-free healthy subjects (mean ± SD: 14.5 ± 1.7 pmol/L versus 21.3 ± 4.6 pmol/L and 142.3 ± 22.7 pmol/106 PBMCs versus 359.3 ± 31.6 pmol/106 PBMCs in plasma and PBMCs, respectively) [22].

Peptidergic Neurotransmitters

5-HT and GABA are neurotransmitters involved in pain modulation and central sensitization at the trigeminal level. In patients with TTH, evidence on peripheral 5-HT levels has been inconsistent. One study found significantly lower platelet 5-HT levels in 13 patients during interictal periods compared with 10 healthy controls (mean ± SEM: 390.3 ± 54 ng/109 platelets versus 545.0 ± 53 ng/109 platelets) [23]. Another study found significantly lower platelet 5-HT levels in 23 patients with muscle contraction headache during interictal periods compared with 20 healthy controls [24]. A third study found significantly lower platelet 5-HT levels in a cohort of 30 patients compared with 30 matched controls (mean ± SD: 361.3 ± 42.4 ng/109 platelets versus 444.5 ± 67.6 ng/109 platelets) [7]. However, these findings were not replicated in another cohort of 20 patients and 15 controls, where no significant difference was observed (mean ± SEM: 53.3 ± 9.26 ng/108 platelets versus 53.1 ± 6.7 ng/108 platelets) [25]. Similarly, a study involving 23 patients with muscle contraction headache and 26 healthy controls found higher platelet 5-HT levels in the patient group (mean ± SD: 23.4 ± 13.7 ng/mL versus 12.5 ± 7.3 ng/mL) [26]. During headache, platelet 5-HT levels were significantly lower in 31 patients with TTH compared with 27 healthy controls (mean ± SD: 508.0 ± 268.2 ng/109 platelets versus 767.7 ± 333.7 ng/109 platelets) [8]. Additionally, significantly reduced plasma 5-HT levels were reported in a large cohort of 310 patients with chronic TTH relative to 35 healthy controls (mean and range: 310 (92–823) ng/109 platelets versus 474 (125–872) ng/109 platelets) [27]. In another study, although plasma 5-HT tended to be higher in patients, no significant differences were observed in plasma 5-HT and platelet 5-HT between 40 patients with chronic TTH and 40 healthy controls (median and quartiles: 3.7 × 10–9 mol/L (2.5–8.0) versus 3.0 × 10–9 mol/L (1.9–5.7) and 3.9 × 10–18 mol/platelet (3.0–4.8) versus 4.1 × 10–18 mol/platelet (3.5–5.0) in plasma and platelets, respectively) [28]. In a study comparing salivary levels, 48 patients with TTH during active headache exhibited significantly higher 5-HT levels than 43 healthy controls (mean ± SD: 1646 ± 1945 ng/mL versus 450 ± 405 ng/mL) [9]. No significant differences were observed in GABA levels between the two groups (mean ± SD: 16.0 ± 18.3 pmol/mL versus 21.6 ± 22.7 pmol/mL) [9]. In a study conducted in a pediatric department, no significant differences in serum 5-HT levels were found between 7 children with episodic TTH and 17 headache-free healthy subjects (mean ± SD: 90.7 ± 26.2 µg/L versus 96 ± 32.9 µg/L) [22].

Gaseous Neurotransmitters

NO is a gaseous neurotransmitter that has roles at the trigeminovascular level that intersect with both sensory and autonomic systems. In a double-blind, placebo-controlled crossover trial, 16 patients with chronic TTH and 16 healthy controls were randomized to receive intravenous infusion of glyceryl trinitrate (GTN, a NO donor) or placebo for 20 min on two headache-free days separated by at least 1 week [29]. GTN infusion induced significantly more headache in patients with chronic TTH, with a biphasic response resulting in an immediate mild headache and a more pronounced delayed headache occurring at 8 h after infusion [29, 30]. In patients with TTH, there was no difference between GTN and placebo regarding muscle hardness, myofascial tenderness or pressure, and heat pain thresholds [30]. Considering potential biomarkers for GTN-induced headache, plasma levels of CGRP did not differ on GTN versus placebo day in either patients or controls (p = 0.65 and p = 0.48, respectively) [31]. GTN may lead to increased endogenous production of NO by activation of the l-arginine–NO pathway. It is known that NO is synthesized from l-arginine and that the reaction also yields citrulline. Plasma levels of arginine were unchanged in both patients and controls at baseline and 60 min after GTN administration [32]. On the other hand, plasma levels of citrulline increased significantly 60 min after the start of GTN infusion compared with placebo in patients with TTH (p = 0.01) [32].

Autonomic Peptides

Pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and neuropeptide Y are autonomic peptides involved in vasodilation and nociceptive modulation at the trigeminal level. Plasma levels of PACAP did not differ between 106 patients with TTH outside attacks and 50 healthy controls (mean ± SD: 39.96 ± 15.90 pg/mL versus 42.45 ± 13.57 pg/mL) [33]. In the external jugular vein, plasma levels of VIP showed no significant differences between 20 patients with chronic TTH and 20 healthy controls, regardless of whether patients were experiencing a headache or not (median and quartiles: 6 pmol/L (4–7) versus 6 pmol/L (4–6) during headache; 6 pmol/L (5–7) versus 6 pmol/L (4–6) during interictal periods) [6]. Similarly, the same study found no significant differences in plasma levels of neuropeptide Y measured in the cranial circulation between the two groups (mean ± SEM: 121 ± 3 pmol/L versus 113 ± 5 pmol/L during headache; 118 ± 3 pmol/L versus 113 ± 5 pmol/L during interictal periods) [6]. Plasma levels of neuropeptide Y were significantly higher in 20 patients with TTH compared with 15 healthy controls (median and range: 3.5 ng/mL, range: 0.7–6.7 versus 2.5 ng/mL, range: 0.9–6.1), with no difference when compared with levels observed in 51 patients with fibromyalgia [34]. In 15 pediatric patients with episodic TTH, no significant modifications were present in plasma levels of neuropeptide Y between interictal periods and attacks, and no differences were observed with 20 healthy controls when neuropeptide Y levels were assessed in interictal periods (mean ± SEM: 133.2 ± 4.1 pmol/L versus142.2 ± 5.5 pmol/L) [35].

Neurotrophins

Brain-derived neurotrophic factor (BDNF) is an abundant neurotrophin within the central and the peripheral nervous system [36]. It is released from trigeminal ganglion neurons by inflammatory stimuli and vasoactive peptides such as CGRP [37, 38]. In a bicenter prospective study, there was no significant difference in serum BDNF levels collected from the cubital vein between 6 patients with frequent TTH outside headache periods and 22 healthy controls (mean ± SD: 20.97 ± 2.49 ng/mL versus 21.20 ± 5.64 ng/mL) [39].

Discussion

This review describes the complexity and heterogeneity of neurotransmitter alterations in TTH, reflecting a multifactorial pathophysiology in which central sensitization and subtle inflammatory mechanisms may all play a role [40]. Although migraine is mentioned throughout the manuscript, the present review focuses exclusively on studies comparing patients with TTH and healthy controls in order to identify neurochemical alterations that may serve as potential therapeutic targets. This approach was selected to isolate pathophysiological features specific to TTH, rather than to highlight differential diagnostic markers. While some neurochemical markers show promising trends, the current evidence is too inconsistent to identify any single mediator as definitively involved in the pathophysiology of TTH. Among sensory neuropeptides, substance P has been the most consistently associated with TTH. Elevated levels were observed in both platelet and salivary samples during interictal periods and active headache [7–9]. Moreover, its inverse correlation with pressure pain thresholds suggests a role in central sensitization [7]. The consistent elevation of substance P in biological samples raises the possibility of its use as a peripheral biomarker for TTH, especially in relation to pain threshold alterations. Additionally, pharmacological modulation of substance P signaling could represent a future therapeutic avenue, although no such agents are currently approved for headache treatment [41]. In contrast, CGRP, a key peptide in migraine pathophysiology, does not appear to be significantly altered in TTH, across studies involving plasma, CSF, and pediatric populations [10–13]. Unlike migraine, TTH may not rely on CGRP-mediated mechanisms [42]. Regarding endogenous opioids, β-endorphins shows a possible trend toward reduced levels in both plasma and PBMCs of patients with TTH evaluated outside headache periods [7, 17, 18, 22]. However, these findings were not consistently replicated across cohorts [12, 15, 16, 19]. MET appears to follow an opposite trend, with elevated concentrations in both plasma and CSF in patients with TTH [12, 20, 21]. This paradox may reflect compensatory mechanisms within the endogenous opioid system or methodological differences in sample collection and analysis, such as the biological matrix used (e.g., plasma versus cerebrospinal fluid), the timing of sampling relative to headache phases, or the assays employed for peptide quantification. Despite heterogeneity in findings, the consistent observation of altered opioid markers supports the hypothesis of dysfunctional descending pain inhibition in TTH [43, 44]. On the other hand, the role of 5-HT in TTH remains controversial. While some studies report reduced 5-HT levels in plasma and platelets [7, 8, 23, 24, 27], others find no difference or even elevated concentrations in patients [9, 25, 26, 28]. Platelets serve as a major peripheral storage site for serotonin and reflect systemic serotonergic tone, although they do not synthesize serotonin themselves. Interestingly, salivary 5-HT was found to be increased during headache attacks in one study [9]. Despite some inconsistencies across studies, the majority of findings (particularly those assessing platelet and plasma 5-HT) suggest a trend toward reduced serotonin levels in individuals with TTH. However, this pattern lacks the consistency and robustness required to support serotonin as a reliable biomarker or therapeutic target in clinical practice. The role of 5-HT in migraine is more clearly established, with low interictal plasma 5-HT levels and a marked release during attacks being well documented [45]. GABA, a key inhibitory neurotransmitter, has been less frequently studied, and current data do not support a significant alteration in its levels in patients with TTH [9]. A human provocation study with GTN suggests that NO plays a role in triggering headache in TTH [29]. Patients receiving GTN experienced a biphasic headache response, indicating a heightened sensitivity to NO-mediated vascular or nociceptive activation. These effects appeared to be CGRP-independent, as CGRP levels remain unchanged after GTN infusion [31]. Beyond CGRP-independent vascular effects, nitric oxide may contribute to headache in TTH by facilitating central sensitization, enhancing glutamatergic transmission [46]. Additionally, NO can modulate nociceptive signaling through increased oxidative stress and direct activation of second messenger systems, such as cyclic GMP, within the trigeminovascular network [47, 48]. These mechanisms could explain the delayed and prolonged headache response observed after GTN infusion in patients with TTH. Nitric oxide-related pathways may support the rationale for targeted interventions that modulate NO synthesis and nonspecific treatments such as antidepressants, which influence monoaminergic and peptidergic systems [49, 50]. Autonomic neuropeptides such as PACAP, VIP, and neuropeptide Y have been extensively studied in migraine, but their role in TTH appears limited [51]. No significant differences in plasma levels between patients and controls have been observed, regardless of headache state [6, 33, 35]. An exception is one study showing elevated neuropeptide Y in patients with TTH compared with healthy individuals, although findings were not consistent across other cohorts [34]. Overall, the data suggest a minimal contribution of the parasympathetic system in TTH pathogenesis. Only one study assessed BDNF, and it found no significant differences in serum levels between patients with TTH and controls [39]. Although BDNF plays a known role in synaptic plasticity and central pain modulation, current evidence does not support its involvement in TTH.

Study Limitations

The present review is limited by substantial heterogeneity across studies, including variations in design, small sample sizes, and differences in the type and handling of biological specimens, as well as in analytical methodologies. The lack of longitudinal data and mechanistic interventional studies further constrains the ability to infer causal relationships or assess treatment responsiveness. In addition, several potential confounding factors such as the use of symptomatic or prophylactic medications and the presence of psychiatric or somatic comorbidities were not consistently reported or controlled for across studies, which may have influenced the observed neurotransmitter alterations. Nevertheless, recurrent findings—such as elevated substance P, dysregulation of endogenous opioids, and nitric oxide-related mechanisms—highlight relevant neurobiological pathways that warrant further targeted investigation in individuals with TTH.

Conclusions

This systematic review did not identify any single neurotransmitter or neuropeptide as a definitive biomarker or central mediator in the pathophysiology of TTH. The available evidence is fragmented and often inconsistent, reflecting methodological heterogeneity and likely pathophysiological complexity. Nonetheless, substance P, endogenous opioids, and nitric oxide have emerged more frequently across studies and may represent promising targets for further investigation. These findings may inform the design of future mechanistic studies and early-phase trials. In particular, pharmacological approaches aimed at modulating the endogenous opioid system or blocking substance P signaling could be explored. Additionally, nonpharmacological strategies such as acupuncture and biofeedback, which are known to affect neuropeptide dynamics, deserve renewed attention in light of their potential neurobiological impact.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 75 KB) (74.6KB, pdf)

Author Contributions

Lanfranco Pellesi, Wei Wang, and Paolo Martelletti conceived the idea for the article. Lanfranco Pellesi, Aidin Yangjeh, Ibrahim Hajjaj, Mousbah Lababidi, and Fezan Sarwar performed the literature search and data analysis. All authors contributed to drafting the manuscript and critically revised the work for important intellectual content. All authors approved the final version of the manuscript.

Funding

No funding or sponsorship was received for this study or publication of this article.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Declarations

Conflict of Interest

Lanfranco Pellesi serves as a member of the Editorial Board of The Journal of Headache and Pain, BMC Neurology, Pain Research and Management, and European Journal of Medical Research. In addition, Lanfranco Pellesi is Editor-in-Training for Clinical and Translational Science. Wei Wang serves as member of the Editorial Board of The Journal of Headache and Pain and SN Comprehensive Clinical Medicine. Paolo Martelletti is the Editor-in-Chief of The Journal of Headache and Pain and of SN Comprehensive Clinical Medicine. Wei Wang and Paolo Martelletti are members of the Editorial Board of Pain and Therapy. Wei Wang and Paolo Martelletti have not been involved in the selection of peer reviewers for the manuscript nor any of the subsequent editorial decisions. Aidin Yangjeh, Ibrahim Hajjaj, Mousbah Lababidi, and Fezan Sarwar declare that they have no competing interests.

Ethical Approval

This study is based on previously conducted studies and does not contain any new studies with human participants or animals performed by any of the authors. For this reason, ethical approval is not required.

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