Abstract

BACKGROUND/AIMS

Headache disorders, particularly migraine and chronic tension-type headache (CTTH), are highly prevalent neurological conditions contributing substantially to global disability. Hematologic and inflammatory markers have been proposed as potential indicators of migraine, yet findings remain inconsistent. This study aimed to examine sex-, age-, and subtype-specific differences in hematologic and inflammatory indices among patients with migraine and CTTH, and healthy controls.

MATERIALS AND METHODS

We conducted a retrospective cross-sectional case-control study in the neurology outpatient clinic between December 2024 and March 2025. Medical records and same-day laboratory results for patients diagnosed with migraine (chronic or episodic) or with CTTH were reviewed. Participants were classified according to the International Classification of Headache Disorders, 3^rd edition criteria. Complete blood counts were used to calculate the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and composite indices, including red cell distribution width to mean corpuscular volume ratio (RDW/MCV), platelet distribution width to platelet count ratio, and hemoglobin (Hb) to RDW ratio.

RESULTS

A total of 1,074 participants were included in the study. Compared with CTTH and controls, migraine patients had lower Hb and hematocrit levels, and higher RDW, NLR, and PLR values (all p<0.01). In the migraine subgroup analysis, patients with aura had lower lymphocyte counts and higher NLR compared with those without aura (p<0.001). Patients with chronic migraine had higher platelet counts and higher Migraine Disability Assessment scores (38.5 vs. 16; p<0.001).

CONCLUSION

Migraine is associated with modest but consistent alterations in hematologic and inflammatory markers, particularly elevated RDW and NLR, supporting systemic immune-vascular involvement and the potential of these markers as adjunctive biomarkers.

INTRODUCTION

Headache disorders are among the most common neurological conditions worldwide, and they impose substantial burdens on individuals and societies. Migraine is a disabling, recurrent headache disorder frequently involving nausea, photophobia, phonophobia, and aura.¹ Over the past decades, the global impact of migraine has increased, with current estimates indicating over a billion affected people and a rise in disability-adjusted life years.^{2, 3} Tension-type headache (TTH), though generally milder, is more pervasive: many people experience it episodically or recurrently during their lifetimes.

A crucial step in tailored treatment and prevention is understanding the biological causes and epidemiology of these headache disorders. One of the most consistent epidemiological features of migraine is its pronounced female preponderance. The prevalence of migraines in women is roughly two to four times higher than in men after puberty.⁴ Women typically report longer attack durations, more frequent recurrences, and greater disability.⁵ Hormonal fluctuations, especially in estrogen, frequently influence migraine risk and severity, impacting the trigeminovascular, vascular, and neural pathways.^{4, 6, 7} By contrast, TTH demonstrates a more uniform sex distribution in some cohorts; despite mixed data, certain studies report a slight male predominance or equal distribution.^{8, 9}

Biological and hematologic factors may underlie the sex- and age-related differences observed in headache disorders. It is well known that males tend to have higher hemoglobin (Hb) and hematocrit levels than females, reflecting physiological sex differences in erythropoiesis and blood volume.¹⁰ On the other hand, females often exhibit elevated red cell distribution width (RDW) and platelet count, which may be related to differences in hematologic regulation, iron metabolism, and microvascular dynamics.¹¹ Baseline sex-based differences in hematologic metrics should be considered when exploring links between these metrics and headache disorders.

Researchers have been investigating associations between hematologic or inflammatory biomarkers and headache phenotypes. Peripheral blood ratios-such as neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and derived composite indices-are useful because they are inexpensive, readily available, and may reflect systemic inflammatory balance. Several studies have examined their links to migraine, albeit with inconsistent findings.^12-15 For example, Sarıcam¹³ found that PLR and neutrophil-monocyte ratio were higher in the migraine group than in the control group, whereas differences in NLR were not significant among the subgroups. Likewise, Gül et al.¹⁵ found that RDW was notably elevated in participants with migraine and that it remained an independent predictor after multivariate adjustment. Moreover, in emergency settings, some studies have evaluated whether NLR or PLR can help distinguish migraine from nonspecific headaches.^{16, 17}

However, findings in the literature remain inconsistent. Some population-based studies did not find robust associations between inflammatory cell counts and migraine during interictal periods, suggesting that attack timing, prophylactic therapy, or selection biases might have influenced the results.¹⁸ Moreover, numerous studies are limited by small sample sizes, lack of subgroup stratification (e.g., by aura or chronicity), or insufficient control for confounders.

In light of this complex background, this study aimed to examine a broad panel of hematologic and inflammation-related indicators across diagnostic groups [migraine, chronic tension-type headache (CTTH), and healthy controls].

By combining demographic, hematologic, and inflammatory data, we aimed to contribute to the evolving understanding of migraine phenotypes, risk stratification, and the pathophysiologic role of systemic immune-vascular interaction.

MATERIALS AND METHODS

This retrospective, cross-sectional, case-control study included 1,074 adults (203 males and 871 females) who attended the neurology outpatient clinic between December 2024 and March 2025. Medical records and same-day laboratory results of patients diagnosed with migraine (chronic or episodic) or CTTH who had visited the clinic at least three times within the previous 12 months were reviewed. The study participants were adults aged 18-65 who met the International Classification of Headache Disorders-3 (ICHD-3) (1) criteria for either migraine or CTTH. Migraine patients were further subdivided into episodic and chronic migraine groups based on attack frequency, and into subgroups with or without aura based on clinical features. The control group comprised individuals with no history of primary headache disorders or chronic pain disorders. All participants were clinically stable, were in an interictal period lasting at least 72 hours before blood sampling, and had not used corticosteroids, non-steroidal  anti-inflammatory drugs, triptans, and other anti-inflammatory/antiplatelet drugs for at least two weeks. The control group consisted of individuals who presented to the same outpatient clinics for non-headache-related complaints during the study period. Controls had no history of primary headache disorders or chronic pain conditions. To ensure comparability, the same exclusion criteria that were applied to the migraine and CTTH groups-including systemic inflammatory diseases, acute or chronic infections, hematological disorders, malignancy, and recent use of medications affecting inflammatory parameters-were also applied to the control group.

Participants were excluded if they had systemic inflammatory or autoimmune diseases, hematological or oncological disorders, chronic systemic illnesses (such as diabetes, renal, hepatic, thyroid, or cardiovascular disease), or any acute infection within the preceding four weeks. Pregnant or breastfeeding women, active smokers, individuals who use alcohol or other substances, and those on anticoagulants or hormonal therapy were not included. Individuals who, in the past three months, underwent surgery, experienced trauma, or received blood transfusions, as well as those with incomplete clinical or laboratory data, were not included in the analysis.

Ethics Approval and Consent to Participate

The University of Health Sciences Türkiye, İzmir City Hospital Non-Interventional Ethics Committee (approval number: 2024/232, date: 04.12.2024) approved the study, which was conducted in accordance with the Declaration of Helsinki. All participants provided written informed consent.

Data Collection and Clinical Assessment

Demographic characteristics, including age and sex, were recorded for all participants. Structured interviews and medical records provided clinical details, such as migraine type, presence of aura, and headache severity. Aura status was determined according to ICHD-3 criteria and recorded from neurologist documentation in the outpatient clinic notes and from structured patient interviews. Headache severity and migraine-related disability were assessed using the visual analog scale and the Migraine Disability Assessment (MIDAS) questionnaire,¹⁹ respectively.

Hematological Measurements

All participants provided venous blood samples after at least eight hours of fasting, during the interictal period for those with migraine or CTTH. The blood samples were assessed via an automated hematology analyzer (Sysmex XN-1000, Kobe, Japan) within 60 minutes of collection. The following parameters were measured: Hb, hematocrit, mean corpuscular volume (MCV), RDW, platelet count, and platelet distribution width (PDW). White blood cell counts, including neutrophil and lymphocyte counts, were also recorded.

From these primary parameters, several inflammatory and composite indices were calculated:

• NLR = Neutrophil count/lymphocyte count

• PLR = Platelet count/lymphocyte count

• The RDW/MCV, PDW/PLT, RDW/PLT, and Hb/RDW were computed to assess variability in erythrocyte and platelet distributions.

Statistical Analysis

Statistical analyses were conducted using R software version 4.4.2 (R Foundation for Statistical Computing, Vienna, Austria). Normality of continuous variables was assessed via the Shapiro-Wilk test. Depending on the data distribution, Student’s t-test or the Wilcoxon rank-sum test was used to compare two independent groups, and one-way ANOVA or the Kruskal-Wallis test was used to compare more than two groups. Categorical variables were analyzed using the chi-square test or Fisher’s exact test when cell counts were fewer than five. In addition to univariable analyses, a multivariable logistic regression analysis was performed to identify independent factors associated with migraine. Variables showing a p-value <0.10 in univariable analyses and clinically relevant covariates were entered into the multivariable model. Age, sex, RDW, NLR, and PLR were included in the final model. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Model fit was assessed using the omnibus chi-square test, Nagelkerke R², and the Hosmer-Lemeshow goodness-of-fit test. Statistical significance was determined by a p-value less than 0.05.

RESULTS

The study included 1,074 subjects; 203 were male (18.9%) and 871 were female (81.1%), with a mean age of 41.5±10.9 years. The distribution of diagnostic groups differed significantly by sex (p<0.001). As shown in Table 1, migraine was more prevalent among females (55.9%), whereas CTTH was more frequent among males (40.8%). Males exhibited significantly higher Hb and hematocrit, while females had higher RDW and platelets (all p<0.001). MCV also differed between sexes (p=0.011). Sex-related differences were observed in several hematological indices, including PLR, RDW/MCV, PDW/PLT, and Hb/RDW ratios (all p<0.001).

When participants were categorized by headache type (Table 2), age and sex distributions differed significantly among groups (p<0.001 for both). CTTH patients were older (47.8±9.5 years) than the migraine group (38.1±9.7 years) and the control group (39.3±11.6 years). The migraine group was predominantly female (89.0%). whereas CTTH had a higher proportion of males (23.9%). Compared with other groups, migraine patients had lower Hb and hematocrit (p<0.001) and higher RDW (p=0.005). Neutrophil and lymphocyte counts differed significantly across groups (p<0.001). Accordingly, NLR values were significantly higher in the migraine group (p<0.001). PDW values were lowest among CTTH patients (p<0.001), while the migraine group had the highest PLR (p=0.002). RDW/MCV and Hb/RDW ratios showed statistically significant differences between groups (p=0.046 and p<0.001, respectively).

In the comparison between episodic and chronic migraine subgroups (Table 3), demographic and hematological parameters were largely similar The MIDAS score was significantly higher in the chronic migraine group (38.5 vs. 16, p<0.001). The chronic migraine group also had a higher platelet count (p=0.047). In addition, the PDW/PLT ratio differed between subtypes (p=0.036).

As shown in Table 4, a comparison of migraine patients with and without aura revealed that those with aura were slightly younger (36.8 vs. 38.3 years; p=0.251) and had significantly lower lymphocyte counts and percentages (both p<0.001). NLR values were significantly higher in patients with aura (p<0.001). These findings suggest a distinct inflammatory balance in aura-associated migraine.

When the study population was stratified by age (Table 5), participants aged >40 years had a higher prevalence of CTTH (44.1% vs. 18.2%; p<0.001) than those aged ≤40 years, while migraine was more frequent in the younger group (62.3% vs. 41.5%; p<0.001). Hb levels were slightly higher in participants aged ≤40 years (p=0.059), whereas RDW was significantly greater among those aged >40 years (p<0.001). PDW/PLT ratios were higher in participants aged ≤40 years (p=0.028), whereas Hb/RDW ratios were lower in participants aged >40 years (p=0.001).

As shown in Table 6, univariable logistic regression analysis demonstrated that female sex (OR =4.03, 95% CI: 2.68-6.09, p<0.001), higher NLR (OR  =1.40, 95% CI: 1.15-1.74, p=0.001), and higher PLR (OR =1.00, 95% CI: 1.00-1.01, p=0.018) were associated with migraine. In the multivariable model, female sex remained a strong independent predictor of migraine (OR =4.04, 95% CI: 2.63-6.22, p<0.001). Additionally, higher NLR was independently associated with migraine after adjustment (OR=1.32, 95% CI: 1.08-1.67, p=0.012). RDW and PLR did not retain statistical significance in the adjusted analysis. The overall model demonstrated an acceptable goodness-of-fit (Hosmer-Lemeshow χ² =10.304, p=0.244) with a Nagelkerke R² of 0.114.

DISCUSSION

The study included 1,074 participants, and we observed consistent differences in demographics, hematologic, and inflammatory indices when considering sex, headache diagnosis (migraine, CTTH, controls), migraine subtypes, and age groups. The results add to the existing literature on primary headache heterogeneity and highlight peripheral hematologic markers as potential correlates of underlying biological processes. Overall, migraine was associated with subtle but consistent alterations-particularly elevated RDW and NLR-which may reflect low-grade inflammatory or oxidative processes. Variations in these disturbances by sex, aura status, and age may indicate biologically heterogeneous presentations of migraine. In this study, multivariable logistic regression analysis identified female sex and elevated NLR as independent factors associated with migraine, whereas RDW and PLR were no longer statistically significant after adjustment. The strong association with female sex is consistent with extensive epidemiological evidence demonstrating a marked female predominance in migraine, likely mediated by hormonal and neurovascular mechanisms.^{4, 20}

A prominent observation was that migraine showed female predominance (55.9%), whereas CTTH was more frequent in males (40.8%). This is consistent with epidemiological research showing that migraines predominantly affect women, particularly after puberty, with a female-to-male ratio of 2:1-3:1.^{4, 20, 21} Hormonal fluctuations, especially estrogen-related mechanisms, may modulate vascular and inflammatory pathways involved in migraine.⁶ In the American Migraine Prevalence and Prevention cohort, Buse et al.²⁰ confirmed that sex is a crucial factor in migraine biology, as they demonstrated sex-based differences in prevalence and disability. Our data show that composite indices (PLR, RDW/MCV, PDW/PLT, Hb/RDW) differ by sex; thus, biomarker interpretation must be sex-specific. Stratified analyses by hormonal status, aura, and comorbidities are crucial for defining biologically meaningful migraine phenotypes.

Migraine patients showed lower Hb and hematocrit levels but higher RDW, NLR, and PLR compared with CTTH and controls; PDW was lowest in CTTH. These findings may point to subtle systemic alterations related to subclinical inflammatory or oxidative mechanisms.²² Similarly, according to Özdemir et al.,¹² patients with TTH also present with increased NLR and PLR values compared with controls, suggesting that systemic inflammatory processes may be a factor in migraine and CTTH. The value of peripheral immune markers, including NLR, PLR, and C-reactive protein (CRP), is supported by their findings in differentiating primary headache disorders and in assessing inflammatory load.¹² The mechanisms behind increased RDW include cytokine-driven suppression of erythropoiesis or the release of immature red blood cells (anisocytosis); oxidative stress or iron dysregulation may exacerbate red cell heterogeneity.^{21, 23} Higher NLR values may reflect a relative predominance of neutrophil counts, which has been interpreted as a marker of systemic inflammatory balance in previous studies.

RDW, which quantifies red cell size variability, has been studied as a marker of inflammation and oxidative stress.^{23, 24} Previous studies demonstrated that those with migraines had higher RDW, with RDW remaining an independent predictor after adjustment.^{5, 25} Our findings are consistent with previous reports and further demonstrate differences in RDW not only between migraine patients and controls, but also between migraine patients and CTTH patients. To clarify temporal dynamics, longitudinal studies could assess RDW and related indices across ictal-interictal periods and during migraine progression (from episodic to chronic). Combining complete blood count (CBC)-derived measurements with cytokines [interleukin (IL)-1 beta, IL-6, tumor necrosis factor-alpha, high-sensitivity CRP] or endothelial function tests may provide greater insight into inflammatory-vascular interactions.

The observation of higher NLR values in migraine is consistent with hypotheses suggesting a role for innate immune mechanisms. The neutrophil-to-lymphocyte axis reflects systemic inflammation and is a prognostic factor in multiple neurological diseases.^{16, 24} However, its value outside of acute episodes is uncertain, since NLR may vary depending on the timing of attacks or comorbidities.¹⁶ Future trials might assess whether anti-inflammatory or antioxidant treatments alter these hematologic signatures and reduce the attack burden.

Platelet indices offer complementary insight. The release of serotonin, thromboxanes, and other vasoactive and proinflammatory mediators from platelets affects neurovascular reactivity. Increased PLR might indicate platelet activation alongside lymphocyte suppression. PDW and derived ratios (PDW/PLT) reflect platelet size variability and reactivity. The current study’s finding of higher PLR in patients with migraine is consistent with studies linking platelet activity to migraine development.¹⁴ Conversely, the lower PDW observed in CTTH may indicate a less reactive platelet phenotype. Based on these findings, migraine, compared with CTTH, may be associated with peripheral hematologic patterns compatible with a pro-inflammatory-oxidative profile. Future studies should focus on multivariable biomarker models that incorporate RDW, NLR, and platelet indices and could be enhanced by machine learning to distinguish migraine subtypes and predict therapeutic response.

Within the migraine cohort, episodic and chronic subtypes showed minimal hematologic differences, except that chronic migraine showed higher platelet counts and altered PDW/PLT ratios. Chronic cases had higher MIDAS scores, which indicate a greater disease burden, as expected. The absence of major differences in RDW or NLR may indicate that migraine chronicity is not solely related to an intensification of systemic inflammation but may involve central sensitization and neuroplasticity.²⁶ Combining neuroimaging measures (such as white matter hyperintensities and perfusion) or immunogenomic data with peripheral biomarkers may reveal links between systemic and central mechanisms.

Patients with migraine with aura were younger, had fewer lymphocytes, and had higher NLRs than those without aura. This pattern may be compatible with differences in immune balance in aura-associated migraine, potentially due to lymphocyte redistribution or immune modulation. Previous studies of serial systemic immune inflammation indices showed fluctuating immune profiles in individuals with and without aura, implying immune changes associated with aura rather than static trait differences.²⁶

Age-related patterns further supported biological variability. CTTH was more common in older participants and was associated with a lower Hb/RDW ratio and higher RDW, which may be related to oxidative stress or age-related changes in erythropoiesis. Younger individuals exhibited higher PDW/PLT ratios, which may indicate increased platelet activity. However, NLR and PLR did not change significantly with age, suggesting that the systemic inflammatory balance remains relatively constant in adults. These results emphasize that age affects blood and headache characteristics.

Even though the results were statistically significant, the biomarker differences were small and overlapped between groups. This pattern suggests limited discriminative performance at the individual patient level, despite statistical significance. Accordingly, RDW-, NLR-, and PLR-based indices should be interpreted as adjunctive markers of low-grade inflammatory balance rather than stand-alone diagnostic tools for primary headache classification. Consequently, these indices may not be sufficient as stand-alone diagnostic tools, though they could help indicate inflammation or differentiate migraine subtypes. Their clinical utility likely lies in combination with other parameters within multimodal predictive frameworks. In addition, multiple hematologic indices were examined across several subgroup comparisons without formal adjustment for multiple testing. This increases the possibility of type I error, and therefore the observed associations should be interpreted with caution.

Taken together, these findings further support NLR as a more robust inflammatory marker in migraine than other CBC-derived indices. NLR reflects the balance between innate and adaptive immune responses and has been proposed as a marker of inflammatory burden in migraine, although prior studies have reported heterogeneous results depending on study design and attack timing.^{13, 16, 17} In contrast, RDW and PLR were not independently associated with migraine after adjustment, suggesting that their univariable associations may reflect sex-related hematologic differences or shared inflammatory pathways rather than migraine-specific effects.^{15, 25} Although the explanatory power of the model was modest, these findings highlight NLR as a more robust inflammatory marker in migraine compared with other CBC-derived indices.

Study Limitations

Several limitations must be acknowledged. First, the retrospective design limits causal inference and introduces potential selection and information biases. In addition, a cross-sectional design precludes causal inference; therefore, it remains unclear whether hematologic alterations precede or result from migraine. Longitudinal monitoring across ictal and interictal periods is warranted. Furthermore, CBC indices are non-specific  and are influenced by systemic conditions. Integrating cytokine, vascular biomarker, or endothelial function test measurements could help clarify the links. Future studies incorporating high-sensitivity CRP, cytokine profiles, and endothelial and vascular biomarkers are needed to better characterize the underlying mechanisms and validate whether CBC-derived indices accurately reflect migraine-related inflammatory and vascular pathways.

Additionally, the clinic-based sample may overrepresent individuals who are more symptomatic, thereby limiting generalizability. Inflammatory markers may be affected by the timing of blood sampling. Even with a large sample, the impact was small. Moreover, the substantial overlap between groups limits clinical interpretability and precludes using these indices as diagnostic classifiers in routine practice. Diagnostic precision and prognostic accuracy might be enhanced in future studies through composite biomarker models and advanced statistical methods. Although multivariable regression analysis was performed, residual confounding cannot be entirely excluded, and the model included a limited number of hematologic variables.

CONCLUSION

Migraine patients exhibit small but consistent variations in blood and inflammatory measures, especially increased RDW and NLR, compared with CTTH patients and control subjects. Biological differences in these relationships are influenced by sex, age, aura, and chronicity. Although not diagnostic in isolation, these indices may provide valuable adjunctive insights into migraine pathophysiology. However, given the modest effect sizes and overlapping distributions, these markers should be considered supportive rather than diagnostic. These findings should be considered exploratory and require confirmation in longitudinal studies incorporating multivariable analytical approaches. Future longitudinal and multimodal studies are warranted to validate these hematologic and inflammatory markers as potential clinical tools.