Serum vitamin D levels in chronic versus episodic migraine: A cross-sectional three-arm study of associations with migraine chronicity and musculoskeletal tenderness

Abstract

Background

Migraine chronification is associated with increased disability and central sensitization, yet potentially modifiable metabolic contributors remain incompletely understood. Vitamin D deficiency has been implicated in neuroinflammatory and neuromuscular pathways relevant to migraine, but its relationship with migraine chronicity and objective musculoskeletal tenderness has not been systematically evaluated.

Methods

In this cross-sectional, three-arm observational study, 50 patients with chronic migraine (CM), 50 with episodic migraine (EM), and 50 age- and sex-matched healthy controls were enrolled. Serum 25-hydroxyvitamin D levels were measured, and deficiency was defined as <30 ng/mL. Muscle and bone tenderness were assessed using a standardized 4-point scale. Group comparisons were performed using ANOVA with post-hoc testing. Multivariable logistic and modified Poisson regression models estimated adjusted odds ratios (aOR) and relative risks (aRR). Trend analysis across chronicity (control → EM → CM) and Pearson correlations were conducted.

Results

Vitamin D levels declined progressively across chronicity (CM 18.7 ± 11.1; EM 26.3 ± 11.6; controls 35.7 ± 12.7 ng/mL; p < 0.001). Deficiency was present in 78% of CM, 60% of EM, and 46% of controls. CM was independently associated with deficiency (aOR 3.57; aRR 1.54). A significant linear trend was observed (β = −7.41 ng/mL per step; p < 0.001). Muscle and bone tenderness were significantly higher in CM. Vitamin D levels inversely correlated with muscle tenderness (r = −0.41, p < 0.001).

Conclusions

Vitamin D deficiency is associated with CM and increased musculoskeletal tenderness. Given the cross-sectional design, causality cannot be inferred, and reverse causation remains a plausible explanation. It warrants further longitudinal and interventional investigation.

Graphical abstract

This is a visual representation of the abstract.

Keywords

chronic migraine vitamin D deficiency vitamin D migraine chronification nutritional factors

Introduction

Migraine is a highly prevalent and disabling neurological disorder characterized by recurrent headache attacks and a range of associated symptoms.¹ Chronic migraine (CM), defined by high-frequency headache occurring on ≥15 days per month, represents a more severe phenotype compared with episodic migraine (EM) and is associated with greater disability, comorbidity burden, and healthcare utilization.^2,3 However, migraine is increasingly recognized as a spectrum disorder, with headache frequency varying over time rather than existing as a fixed categorical state. Furthermore, headache frequency may fluctuate over time, and the distinction of migraine as episodic or chronic represents a temporal classification rather than a fixed disease state.⁴ Although the pathophysiology of migraine involves complex interactions between genetic, neurovascular, inflammatory, and central sensitization mechanisms, potentially modifiable metabolic factors have gained increasing attention.⁵

Vitamin D, a secosteroid hormone with pleiotropic effects, plays an important role in immune modulation, inflammatory pathways, neuromuscular function, and pain processing. Vitamin D receptors are widely expressed in the central nervous system, including cortical, thalamic, hypothalamic, and periaqueductal gray regions involved in nociceptive processing and relevant to migraine pathophysiology.^6,7 Emerging evidence suggests that hypovitaminosis D may contribute to enhanced nociceptive sensitivity, musculoskeletal pain, and central sensitization, mechanisms that are particularly relevant in CM.⁸ Several studies have reported a high prevalence of vitamin D deficiency (<20 ng/mL) or insufficiency (20–29.9 ng/mL) among patients living with migraine; however, findings remain inconsistent, and comparative data between CM, EM, and healthy controls are limited.^9,10 Moreover, the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and objective measures of musculoskeletal tenderness in headache patients has not been systematically explored. Muscular and bone tenderness are clinically relevant markers of vitamin D deficiency that may reflect heightened pain sensitivity and central sensitization in migraine.^11,12

Understanding whether vitamin D deficiency is differentially associated with migraine chronicity and musculoskeletal tenderness may provide insight into potential pathophysiological links and therapeutic implications. Therefore, the present study aimed to compare serum 25(OH) vitamin D levels among patients with CM, EM, and healthy controls, to evaluate the prevalence and risk of vitamin D deficiency across groups, and to examine the association between vitamin D levels and muscle and bone tenderness scores.

Methods

Study design and setting

This was a cross-sectional, three-arm observational study conducted at a tertiary care neurology outpatient clinic between March 2025 and December 2025. The objective was to compare serum 25-hydroxyvitamin D [25(OH)D] levels among patients with EM, CM, and age- and sex-matched healthy controls. The study protocol was reviewed and approved by the institutional ethics committee, and written informed consent was obtained from all participants before enrollment. The research was executed in line with the principles of the Declaration of Helsinki. This study is reported in accordance with the STROBE guidelines for an observational study.

Study population

Adult patients (>18 years) with a diagnosis of EM or CM were consecutively recruited from the neurology clinic. The diagnosis was established according to the International Classification of Headache Disorders, 3rd edition (ICHD-3) criteria. CM was defined as a headache occurring on at least 15 days per month for more than three months, with at least eight days per month fulfilling criteria for migraine. EM was defined as migraine occurring on fewer than 15 days per month.

Control subjects were recruited from individuals accompanying patients to the hospital for complaints unrelated to headache. Control participants were screened using a structured clinical interview based on ICHD-3 criteria to exclude both current and prior primary headache disorders. Individuals with any history suggestive of migraine, tension-type headache, or other primary headache syndromes were excluded. In addition, individuals reporting occasional headaches related to acute illness, alcohol intake, or other non-specific triggers were also excluded to minimize the inclusion of participants with potential underlying migrainous biology.

Participants were excluded if they were currently receiving vitamin D supplementation or had medical conditions known to affect vitamin D metabolism, including chronic kidney disease, chronic liver disease, malabsorption syndromes, endocrine disorders affecting calcium metabolism, autoimmune or inflammatory disorders, recent infection, recent hospitalization, or pregnancy.

Sample size calculation

Based on preliminary data suggesting a large ANOVA effect size (Cohen's f approximately 0.45–0.50), with a two-sided alpha level of 0.05 and statistical power of 80%, the minimum total sample size required was approximately 135 participants, corresponding to approximately 45 participants per group. To increase statistical precision and allow for multivariable and subgroup analyses, 150 participants were enrolled, including 50 individuals in each group. Post-hoc power analysis demonstrated greater than 99% power to detect between-group differences in mean serum vitamin D levels.

Estimation of serum vitamin D levels

Venous blood samples were collected from all participants under standardized conditions. Serum was separated and analyzed for 25-hydroxyvitamin D [25(OH)D], which is the accepted biomarker of vitamin D status. The procedure was performed according to the instructions provided by the manufacturer (vitamin D enzyme immunoassay kit; Bio-Detect, CA, USA), and results were expressed in ng/mL. Normal vitamin D levels were defined as serum levels >30 ng/mL.

Clinical assessment

All participants underwent a structured clinical evaluation performed by a single neurologist with clinical experience in headache disorders. Demographic data, including age, sex, body mass index (BMI), smoking status, alcohol use, residence, educational level, socioeconomic status, and season of enrollment, were recorded. For patients with migraine, detailed headache characteristics were obtained, including duration of illness in months, headache laterality (unilateral or bilateral), headache quality (throbbing or pressing), headache intensity (moderate or severe), and associated symptoms such as nausea, vomiting, photophobia, and phonophobia. Additional symptoms, including fatigue, limb pain, back pain, generalized body pain, depression, and anxiety, were documented.

Muscle and bone tenderness assessment

As vitamin D deficiency is associated with musculoskeletal pain, muscle tenderness was evaluated using a standardized palpation protocol.^13,14 The following muscle groups were examined bilaterally: temporalis, frontalis, suboccipital region, elbow region, thigh, and Achilles tendon. Tenderness was assessed by applying uniform pressure and graded using a 4-point scale (0–3). A score of 0 indicated no tenderness; 1 indicated a verbal report of mild pain without visible facial reaction; 2 indicated painful tenderness accompanied by a facial expression of discomfort; and 3 indicated severe pain with marked grimacing. The Total Tenderness Score was calculated by summing the individual site scores for each participant. As previous studies have not demonstrated significant side-to-side variability, tenderness from only one side was used for the calculation of the total score.^13,14 Randomization of side selection was not performed, as the clinically predominant side was considered more relevant for assessment. In participants with unilateral migraine, tenderness assessment was performed on the side most frequently affected by headache. If no clear side predominance was identified, the dominant side was used for standardized assessment. Bone tenderness was assessed through palpation of the radius/ulna and anterior tibia. Tenderness was graded using the same ordinal scale as for muscle tenderness.¹⁴ Scores were summed to generate a bone tenderness score, with higher values indicating increased tenderness. Bone tenderness was assessed by palpation of the radius/ulna and anterior tibia, which are clinically accessible sites commonly used in the evaluation of bone tenderness in conditions associated with vitamin D deficiency and osteomalacia.¹⁵ These sites have also been used in previous studies examining musculoskeletal tenderness in headache disorders associated with hypovitaminosis D, particularly in chronic tension-type headache.^14,16

The same neurologist performed all examinations to minimize inter-observer variability. Uniform manual pressure was applied across participants; however, no instrument-based calibration was used. Formal inter-rater reliability testing was not performed. Wherever feasible, participants were assessed during the interictal period to minimize the potential influence of ictal cutaneous allodynia on tenderness assessment.

Statistical analysis

Statistical analyses were performed using Microsoft Excel and GraphPad. Continuous variables were expressed as mean ± standard deviation (SD), and categorical variables were presented as frequencies and percentages. Normality of continuous variables was assessed using the Shapiro–Wilk test. Comparisons of continuous variables across the three groups (EM, CM, and controls) were performed using one-way analysis of variance (ANOVA). When overall group differences were significant, post-hoc pairwise comparisons were conducted using Tukey's honestly significant difference (HSD) test. Effect size for ANOVA was calculated using eta squared (η²), and standardized mean differences for pairwise comparisons were calculated using Cohen's d. For variables that did not meet normality assumptions, the Kruskal–Wallis test was performed as a non-parametric sensitivity analysis. Categorical variables were compared using the chi-square test or Fisher's exact test where appropriate.

The Endocrine Society classifies serum 25(OH) vitamin D levels <20 ng/mL as deficiency and 20–29 ng/mL as insufficiency.¹⁷ Although skeletal complications are most clearly associated with levels <20 ng/mL,¹⁷ several studies suggest that extra-skeletal effects, including neuromuscular and immune modulation, may occur at higher concentrations traditionally classified as insufficient.¹⁸ Given the potential relevance of these mechanisms to migraine pathophysiology, we used a threshold of <30 ng/mL to define suboptimal vitamin D status.

Crude associations between the migraine group and vitamin D deficiency were assessed by calculating relative risks (RR) and odds ratios (OR) with 95% confidence intervals (CI). Multivariable logistic regression analysis was performed to estimate adjusted odds ratios (aOR) controlling for age, sex, and BMI. Because vitamin D deficiency was common in the study population, modified Poisson regression with robust variance estimation was additionally performed to directly estimate adjusted relative risks (aRR), which are more clinically interpretable when outcome prevalence is high. Trend analysis across migraine chronicity (control → EM → CM) was conducted using ordinal coding of group status. Linear regression was used for continuous vitamin D levels, and logistic regression was used for vitamin D deficiency. Correlation between serum 25(OH) vitamin D levels and muscle tenderness scores was assessed using Pearson's correlation coefficient. Linear regression analysis was performed to estimate the regression coefficient (β) and coefficient of determination (R²). Scatter plots with regression lines were generated to illustrate associations. For analyses involving multiple symptom comparisons, p-values were adjusted using the Benjamini–Hochberg false discovery rate (FDR) procedure.

All statistical tests were two-tailed, and a p-value <0.05 was considered statistically significant.

This study was conducted and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines for observational studies.

Results

A total of 64 patients with CM and 61 patients with EM were screened during the study period. Of these, 14 patients with CM and 11 with EM were excluded according to predefined criteria. Reasons for exclusion included age <18 years (n = 2), current vitamin D supplementation (n = 9), presence of medical comorbidities (n = 10; hypertension, n = 4; diabetes mellitus, n = 4; hypothyroidism, n = 2), history of alcohol use (n = 2), and refusal to participate (n = 2). After applying the exclusion criteria, 50 patients with CM and 50 patients with EM were included in the final analysis. In parallel, 50 healthy controls matched for age and sex were recruited during the same study period. Baseline characteristics were comparable across groups, with no significant differences observed in age, sex distribution, BMI, lifestyle factors, educational level, socioeconomic status, or season of enrollment (Table 1).

Table 1.

Main characteristics of episodic migraine, chronic migraine, and controls

Variable	Episodic migraine (n = 50)	Chronic migraine (n = 50)	Controls (n = 50)	p-value
Age (years), mean ± SD	34.24 ± 11.6	36.62 ± 12.1	36.5 ± 11.1	0.56†
Female sex, n (%)	31 (62%)	30 (60%)	27 (54%)	0.68‡
BMI (kg/m²), mean ± SD	24.4 ± 1.9	24.2 ± 1.2	24.2 ± 2.1	0.74†
Smoking, n (%)	6 (12%)	6 (12%)	8 (16%)	0.81‡
Alcohol use, n (%)	5 (10%)	6 (12%)	8 (16%)	0.62‡
Residence, n (%)				0.69‡
Rural	30 (60%)	32 (64%)	34 (68%)
Urban	20 (40%)	18 (36%)	16 (32%)
Education Level, n (%)				0.83‡
≥10 years education	24(48%)	21 (42%)	21 (42%)
<10 years of education	26(52%)	29 (58%)	29(58%)
Socio-economic status				0.94‡
Low	30 (60%)	28 (56%)	29 (58%)
High	20 (40%)	22 (44%)	21(42%)
Season-enrolled				1.00‡
Summer-Fall, n (%)	26(52%)	26 (52%)	26 (52%)
Winter-Spring	24(48%)	24 (48%)	24 (48%)
Serum Vitamin D (ng/mL), mean ± SD	26.27 ± 11.56	18.72 ± 11.1	35.71 ± 12.7	< 0.0001†*
Serum Vitamin D < 30 ng/mL (%)	30(60%)	39 (78%)	23 (46%)	0.003‡
Serum Vitamin D ≥ 30 ng/mL, n (%)	20(40%)	11(22%)	27 (54%)

†One-way ANOVA for continuous variables (age, BMI, serum vitamin D).

‡Chi-square test. Categorical variables (sex, smoking, alcohol, residence, education, SES, season, and frequency of vitamin D deficiency).

*The difference remained statistically significant using the Kruskal–Wallis test (p < 0.001).

Serum vitamin D levels

Serum vitamin D levels differed significantly among the three groups. Mean serum vitamin D concentrations were lowest in patients living with CM (18.72 ± 11.1 ng/mL), intermediate in EM (26.27 ± 11.56 ng/mL), and highest in controls (35.71 ± 12.7 ng/mL). The difference was statistically significant by one-way ANOVA (F(2147) = 17.40, p < 0.001, η²=0.19) and this finding remained significant on non-parametric analysis using the Kruskal–Wallis test (p < 0.001).

Post-hoc pairwise comparisons demonstrated significant differences in serum vitamin D levels between all groups. Patients living with CM had significantly lower vitamin D levels compared with controls (mean difference −16.99 ng/mL, p < 0.001; Cohen's d = 1.42, very large effect). Similarly, individuals living with CM also had significantly lower levels than those with EM (mean difference −7.55 ng/mL, p = 0.003; Cohen's d = 0.67, moderate-to-large effect). Patients with EM had significantly lower vitamin D levels than controls (mean difference −9.44 ng/mL, p = 0.027; Cohen's d = 0.78, large effect) (Table 2).

Table 2.

Post-hoc pairwise comparisons of serum vitamin D levels (Tukey HSD).

Comparison	Mean difference (ng/mL)	p-value	Cohen's d
Chronic vs Control	−16.99	<0.001	1.42
Chronic vs Episodic	−7.55	0.003	0.67
Episodic vs Control	−9.44	0.027	0.78

Values of Cohen's d are reported as absolute magnitude. Effect size interpretation: 0.2 small, 0.5 medium, 0.8 large.

Crude association between migraine status and vitamin D deficiency

Vitamin D deficiency (<30 ng/mL) was observed in 78% of individuals with CM, 60% of EM, and 46% of controls (p < 0.001). In crude analysis, CM was associated with a significantly increased risk of vitamin D deficiency compared with controls (RR 1.56, 95% CI 1.14–2.14) and markedly higher odds (OR 4.16, 95% CI 1.74–9.94). Patients with vitamin D deficiency were 2.36 times more likely to have CM compared to EM (OR 2.36, 95% CI 0.98–5.68). While there was an increased trend (OR = 1.76), the association between vitamin D deficiency and EM alone did not reach statistical significance in this sample size (p = 0.141). In multivariable logistic regression adjusting for age, sex, and BMI, CM remained independently associated with vitamin D deficiency (adjusted OR 3.57, 95% CI 1.47–8.68). In modified Poisson regression, the aRR was 1.54 (95% CI 1.13–2.09), confirming the robustness of the association (Table 3). EM was not independently associated with deficiency even after adjustment. Because vitamin D deficiency was common in this cohort, RRs were more clinically interpreted than OR. Therefore, modified Poisson regression was used to estimate aRRs.

Table 3.

Association between migraine status and vitamin D deficiency (<30 ng/mL).

Comparison	Crude RR (95% CI)	Crude OR (95% CI)	Adjusted OR* (95% CI)	Adjusted RR† (95% CI)	p-value
Chronic vs Control	1.56 (1.14–2.14)	4.16 (1.74–9.94)	3.57 (1.47–8.68)	1.54 (1.13–2.09)	0.006
Episodic vs Control	1.30 (0.92–1.83)	1.76 (0.80–3.89)	1.61 (0.71–3.64)	1.28 (0.91–1.79)	0.14
Chronic vs Episodic	1.20 (0.96–1.50)	2.36 (0.98–5.68)	2.22 (0.91–5.40)	1.20 (0.96–1.50)	0.07

*Adjusted OR from multivariable logistic regression.

†Adjusted RR from modified Poisson regression with robust variance.

Models adjusted for age, sex, and BMI.

Trend analysis across migraine chronicity

A significant linear trend was observed across groups (control → EM → CM). In linear regression analysis, using group status was modelled as an ordinal variable (control = 0, EM = 1, CM = 2) to assess trends across increasing headache frequency categories, serum vitamin D levels decreased by 7.41 ng/mL for each step increase in migraine chronicity (β = −7.41, 95% CI −9.89 to −4.93; p < 0.001). Similarly, logistic regression demonstrated a significant increasing trend in vitamin D deficiency across groups. For each step increase in chronicity, the odds of vitamin D deficiency increased by 89% (OR 1.89, 95% CI 1.22–2.91; p = 0.004) (Table 4).

Table 4.

Trend analysis across migraine chronicity.

Outcome	Estimate per step increase (Control→Episodic→Chronic)	95% CI	p-value
Mean vitamin D (β, ng/mL)	−7.41	−9.89 to −4.93	<0.001
Vitamin D deficiency (OR)	1.89	1.22–2.91	0.004

Migraine chronicity was modeled as an ordinal variable (Control = 0, Episodic = 1, Chronic = 2). Linear regression was used for mean vitamin D levels and logistic regression for vitamin D deficiency (<30 ng/mL). Estimates represent change per step increase in chronicity.

Clinical characteristics of patients living with migraine

Individuals with CM had a significantly longer duration of illness than EM patients (p = 0.002). Bilateral headache was more common in CM (p = 0.021), while headache intensity did not differ significantly between groups (p = 0.18). Associated symptoms such as nausea (p = 0.64), vomiting (p = 0.71), photophobia (p = 0.53), and phonophobia (p = 0.48) were comparable between groups. However, fatigue (p = 0.034), generalized body pain (p = 0.028), limb pain (p = 0.041), and back pain (p = 0.037) were more frequently reported among patients living with CM (Table 5).

Table 5.

Clinical characteristics and musculoskeletal tenderness across study groups.

Variable	Episodic migraine (n = 50)	Chronic migraine (n = 50)	Controls(n = 50)	p-value	Effect size
Headache
Duration of illness	23.76 ± 15.8	22.32 ± 16.9		0.12†	Cohen's d = 0.09
Unilateral headache, n (%)	37 (74%)	29 (58%)		0.02‡	OR ≈ 2.06
Bilateral headache	25 (50%)	39 (78%)		0.021‡	OR ≈ 3.36
Throbbing	30 (60%)	30 (60%)		1.0‡	OR = 1.00
Pressing type	39 (78%)	39 (78%)		1.0‡	OR = 1.00
Moderate	33 (66%)	36 (72%)		0.51‡	OR ≈ 1.32
Severe headache	39 (78%)	41 (82%)		0.62‡	OR = 1.29
Nausea	46 (92%)	46 (92%)		1.0‡	OR = 1.00
Vomiting	16 (32%)	12 (24%)		0.71‡	OR = 0.67
Photophobia	46 (92%)	45 (90%)		0.53‡	OR = 0.79
Phonophobia	43 (86%)	43 (86%)		1.0‡	OR = 1.00
Fatigue	17(34%)	33(66%)	14(28%)	0.034‡	Cramér's V = 0.24
Limb pain	12 (24%)	21 (42%)	9 (18%)	0.041‡	V = 0.22
Backache	18 (36%)	30 (60%)	14(28%)	0.037‡	V = 0.23
Generalized body pain	9 (18%)	22 (44%)	8 (16%)	0.028‡	V = 0.25
Depression	17(34%)	22 (44%)	14(28%)	0.29‡	V = 0.10
Anxiety	10 (20%)	15 (30%)	10 (20%)	0.33‡	V = 0.11
Muscle tenderness score
Total score	3.51 ± 3.3	3.72 ± 3.7	1.32 ± 1.9	< 0.001§	η² = 0.11; d = 0.82*
Temporalis	0.44 ± 0.5	0.98 ± 0.6	0.16 ± 0.4	< 0.001§	η² = 0.15
Frontalis	0.28 ± 0.5	0.25 ± 0.4	0.12 ± 0.3	0.11§	η² = 0.02
Suboccipital	0.24 ± 0.5	0.18 ± 0.4	0.0 ± 0.0	0.018§	η² = 0.05
Elbow	0.31 ± 0.4	0.48 ± 0.5	0.22 ± 0.4	0.021§	η² = 0.05
Thigh	0.16 ± 0.4	0.76 ± 0.5	0.06 ± 0.2	< 0.001§	η² = 0.17
Achilles	0.38 ± 0.3	0.56 ± 0.6	0.10 ± 0.3	< 0.001§	η² = 0.14
Bone tenderness score	1.31 ± 2.1	1.88 ± 2.3	0.8 ± 1.3	0.032§	η² = 0.06; d = 0.59*
Radius-ulna	0.2 ± 0.4	0.31 ± 0.5	0.24 ± 0.4	0.62	η² = 0.01
Anterior tibia	0.46 ± 0.7	0.64 ± 0.8	0.16 ± 0.4	0.004	η² = 0.08

†Independent samples t-test (Episodic vs Chronic).

‡Chi-square test or Fisher's exact test.

§One-way ANOVA (three-group comparison). Non-parametric Kruskal–Wallis testing yielded similar results.

*Cohen's d refers to Chronic versus Control comparison.

Comparison of muscle and bone tenderness scores across groups

Muscle tenderness scores differed significantly across the three groups (p < 0.01), with a moderate overall effect size (η² = 0.11). Both individuals with EM (3.51 ± 3.3) and CM (3.72 ± 3.7) demonstrated significantly higher muscle tenderness scores compared with controls (1.32 ± 1.9). Post-hoc analysis using Tukey's HSD revealed large standardized differences between EM and controls (Cohen's d = 0.77) and between CM and controls (Cohen's d = 0.82). There was no significant difference between CM and EM groups (p = 0.81; Cohen's d = 0.06) (Table 6).

Table 6.

Comparison of muscle and bone tenderness scores across groups.

Variable	Control (n = 50)Mean ± SD	Episodic (n = 50)Mean ± SD	Chronic (n = 50)Mean ± SD	Overall p-value	η²	Pairwise comparison	Pairwise p-value	Cohen's d
Muscle tenderness score	1.32 ± 1.9	3.51 ± 3.3	3.72 ± 3.7	<0.01	0.11	Episodic vs Control	0.01*	0.77
						Chronic vs Control	<0.01*	0.82
						Chronic vs Episodic	0.81	0.06
Bone tenderness score	0.80 ± 1.3	1.31 ± 2.1	1.88 ± 2.3	0.03	0.06	Episodic vs Control	0.18	0.29
						Chronic vs Control	0.02*	0.59
						Chronic vs Episodic	0.24	0.26

Overall comparisons were performed using one-way ANOVA. Post-hoc pairwise comparisons were performed using Tukey's HSD test. η² represents effect size for overall ANOVA. Cohen's d represents standardized mean differences for pairwise comparisons.

*Significant.

Bone tenderness scores also differed significantly across groups (p = 0.03), with a small-to-moderate overall effect size (η² = 0.06). Participants with CM had significantly higher bone tenderness scores (1.88 ± 2.3) compared with controls (0.80 ± 1.3), with a moderate effect size (Cohen's d = 0.59). Differences between EM and controls (Cohen's d = 0.29) and between CM and EM (Cohen's d = 0.26) were not statistically significant. Overall, patients with migraine exhibited significantly greater musculoskeletal tenderness than controls, with the largest effects observed in comparisons involving CM. Linear regression demonstrated a modest but statistically significant inverse association between serum 25(OH) vitamin D levels and total muscle tenderness score (β = −0.19, R² = 0.17). Pearson correlation confirmed this relationship (r = −0.41, p < 0.001) (Figure 1). Lower vitamin D levels were associated with greater muscular tenderness. This association remained evident when stratified by migraine subtype.

Figure 1.

Scatter plot demonstrating the inverse association between serum 25-hydroxyvitamin D [25(OH)D] levels and total muscle tenderness score.

Systemic and affective features associated with vitamin D deficiency

In addition to migraine chronicity and tenderness burden, we observed significantly lower serum 25(OH) vitamin D levels in patients with fatigue, limb pain, and depression. The strongest association was noted for depression, followed by fatigue and limb pain (Table 7). Given multiple comparisons across seven symptom variables, p-values were adjusted using the Benjamini–Hochberg FDR procedure. After FDR correction, the associations between lower serum 25(OH) vitamin D levels and depression (FDR-adjusted p = 0.006) as well as fatigue (FDR-adjusted p = 0.018) remained statistically significant. The association with limb pain did not remain significant after adjustment (FDR-adjusted p = 0.051). No other symptom variables reached statistical significance following correction.

Table 7.

Association between clinical symptoms and serum 25(OH) vitamin D levels.

Symptom	Yes (Mean ± SD)	No (Mean ± SD)	Mean difference (95% CI)	p-value	FDR-adjusted p-value	Cohen's d
Fatigue	19.26 ± 11.7	25.89 ± 11.2	6.63 (2.10–11.16)	0.005	0.018	0.58
Depression	17.81 ± 9.4	25.62 ± 12.4	7.81 (3.36–12.26)	<0.001	0.006	0.71
Limb pain	18.75 ± 9.9	24.47 ± 12.4	5.72 (0.82–10.62)	0.022	0.051	0.49
Back pain	21.11 ± 12.9	23.93 ± 10.8	2.82 (−1.87–7.51)	0.237	0.332	0.24
Generalized pain	19.89 ± 10.8	23.78 ± 12.2	3.89 (−1.13–8.91)	0.126	0.332	0.33
Anxiety	24.27 ± 10.8	22.02 ± 12.3	2.25 (−3.29–7.79)	0.416	0.485	0.19
Bilateral headache	22.18 ± 12.8	23.28 ± 10.1	1.10 (−3.82–6.02)	0.656	0.656	0.09

Values are mean ± SD. Comparisons were performed using independent t-tests. Effect size expressed as Cohen's d. p-values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method.

Discussion

In this three-arm observational study, serum 25-hydroxyvitamin D [25(OH)D] levels declined progressively across migraine chronicity, with the lowest concentrations observed in CM, intermediate levels in EM, and the highest levels in healthy controls. Vitamin D deficiency was independently associated with CM after adjustment for age, sex, and BMI. Trend analysis demonstrated a dose–response relationship across chronicity (control → episodic → chronic), strengthening the biological plausibility of the association. In addition, lower vitamin D levels were moderately associated with greater muscle tenderness, and participants with CM exhibited significantly higher muscle and bone tenderness scores compared with controls. Fatigue, limb pain, and depression were also associated with lower vitamin D levels. Together, these findings suggest that hypovitaminosis D may be linked not only to migraine presence but also to migraine chronicity and systemic musculoskeletal symptom burden. Given the cross-sectional design of this study, causal relationships cannot be established. It remains possible that lower vitamin D levels are a consequence rather than a cause of increased migraine burden.

An important observation in this study was the high prevalence of vitamin D deficiency across all groups, including healthy controls. This finding may reflect population-specific factors such as limited sunlight exposure, dietary habits, or regional variations in vitamin D status. The high baseline prevalence of deficiency may have influenced the magnitude of differences observed between groups and should be considered when interpreting the results.

Most observational studies and meta-analyses report lower mean serum 25(OH)D levels in individuals with migraine compared with controls, although findings are not entirely consistent.¹⁰ Some studies have demonstrated inverse associations between vitamin D levels and monthly migraine days, whereas others have not identified a significant relationship with headache frequency.^9,19 Our findings extend this literature by demonstrating a graded decline in vitamin D levels across migraine chronicity and by estimating both aOR and aRRs. Because vitamin D deficiency was common in our cohort, modified Poisson regression was used to provide clinically interpretable RR estimates. The persistence of association across multiple modelling strategies suggests statistical robustness. However, the cross-sectional design precludes causal inference. Reverse causation is a plausible explanation for the observed association. Individuals with CM may have reduced sunlight exposure due to photophobia, fatigue, and reduced outdoor activity, which may contribute to lower vitamin D levels.

Musculoskeletal tenderness, chronic migraine, and vitamin D deficiency: Any overlap?

A key observation of this study is the inverse association between serum vitamin D levels and cephalic and extracephalic muscle tenderness. Increased muscle tenderness, trigger points, and muscle hardness are well-recognized features of chronic tension-type headache and are considered contributors to sustained peripheral nociceptive input.²⁰ Although migraine is classically conceptualized as a centrally driven disorder involving cortical and brainstem dysfunction, increasing evidence suggests that peripheral input from pericranial and cervical structures may modulate attack frequency and chronification. Increased muscle tenderness has also been reported in migraine, with some studies showing comparable pericranial and cervical tenderness scores in chronic tension-type headache (CTTH) and CM.²¹ Anxiety and combined anxiety–depression have been associated with higher tenderness scores in migraine.²² Although anxiety and depressive symptoms were more common in patients with migraine in our cohort, these differences did not reach statistical significance. The interpretation of increased muscle tenderness requires caution. Migraine, particularly CM, is frequently associated with cutaneous allodynia, which can manifest as tenderness in cephalic and cervical regions. Distinguishing muscle tenderness from cutaneous allodynia may be challenging, particularly in migraine, where allodynia can vary between ictal and interictal states. This may have influenced the assessment of tenderness and represents a potential source of measurement bias. In addition, migraine is associated with higher rates of comorbid pain conditions such as fibromyalgia,²³ which may independently contribute to musculoskeletal tenderness. The higher frequency of generalized body pain observed in the CM group in our study further supports this possibility. Therefore, the observed tenderness may not be exclusively attributable to vitamin D deficiency or peripheral myofascial mechanisms. Accordingly, the relationship between vitamin D levels and tenderness should be interpreted cautiously, as cutaneous allodynia and comorbid pain syndromes may act as confounding factors.

Another important observation of this study is the inverse association between serum vitamin D levels and bone tenderness. To our knowledge, bone tenderness has not been systematically evaluated in migraine populations. However, previous studies in CTTH have reported bone tenderness in association with vitamin D deficiency.^14,16 Vitamin D deficiency is known to contribute to musculoskeletal pain through impaired mineralization, periosteal nociceptor activation, and altered neuromuscular function, which may plausibly help explain the increased bone tenderness observed in our cohort.^17,24

Taken together, these findings suggest that hypovitaminosis D may be linked not only to the presence of migraine but also to migraine chronification and associated musculoskeletal tenderness. The overlap in tenderness patterns between CTTH and CM further supports the hypothesis of a shared musculoskeletal–sensitization axis that may be associated with persistent headache phenotypes.

Systemic and affective features, chronic migraine, and vitamin D deficiency: Any overlap?

In our cohort, fatigue, limb pain, backache, generalized body pain, depression, and anxiety were more frequent in CM. Although only depression and fatigue remained significantly associated with lower vitamin D levels, limb pain, backache, and generalized body pain were more frequent in CM. While CM is increasingly recognized as a systemic pain disorder characterized by widespread somatic hypersensitivity, these same clinical features are well-established manifestations of vitamin D deficiency.^25,26 The convergence of these symptoms raises the possibility of a shared pathophysiological substrate rather than simple clinical coincidence. In this framework, hypovitaminosis D may act as a permissive or amplifying factor that intensifies somatic symptom burden and facilitates migraine chronification in susceptible individuals.

Pathophysiology

Epidemiological evidence demonstrates a strong bidirectional association between chronic daily headache and musculoskeletal symptoms, suggesting that each condition may predispose to or perpetuate the other.²⁷ Generalized musculoskeletal pain in chronic headache disorders is commonly attributed to central sensitization. Vitamin D deficiency is also strongly associated with musculoskeletal pain syndromes. This overlap raises an important mechanistic question: does hypovitaminosis D independently generate musculoskeletal pain in headache patients, does it modify headache pathophysiology, or do both mechanisms interact synergistically?

Peripheral mechanisms in primary headaches

The pathophysiology of primary headache disorders reflects a dynamic interplay between peripheral and central mechanisms. In tension-type headache, peripheral mechanisms are considered predominant and include myofascial factors such as pericranial muscle tenderness, increased muscle hardness, and the presence of myofascial trigger points. In migraine, central mechanisms are generally regarded as the primary drivers of attacks, involving cortical hyperexcitability, brainstem dysfunction, and sensitization within the trigeminovascular system.⁵ Peripheral mechanisms in migraine are largely related to activation of the trigeminovascular pathway. The contribution of myofascial factors to migraine pathophysiology remains less clearly defined. Cutaneous allodynia, a common feature in migraine, is considered a clinical marker of central sensitization.²⁸

The presence of increased muscle tenderness, muscle hardness, the presence of trigger points and bone tenderness in our CM cohort suggests that peripheral myofascial mechanisms may also play a contributory role in migraine chronification. These findings support the possibility that, in a subset of patients, migraine chronification may involve a greater contribution of peripheral myofascial mechanisms than traditionally recognized.

Vitamin D deficiency affecting musculoskeletal in headaches

Vitamin D deficiency has been linked to impaired mineral metabolism, type II muscle fiber atrophy, altered neuromuscular transmission, and increased nociceptor excitability. Selective atrophy of type II fibers increases mechanical load on the remaining fibers, promoting localized ischemia, metabolic stress, and sustained contracture.²⁴ These changes predispose to the formation of myofascial trigger points—features classically described in tension-type headache.¹⁴ Experimental models suggest that vitamin D deficiency may promote sensory hyperinnervation and muscular hypersensitivity.²⁹ Defective mineralization results in the accumulation of unmineralized osteoid along the periosteal surface. This swollen osteoid exerts outward pressure on the periosteum, a structure richly innervated by nociceptive fibers.²⁴ These may also cause musculoskeletal pain. The increased bone tenderness observed in our cohort may therefore reflect subclinical disturbances in mineral metabolism in a subset of patients.

Central modification of migraine by vitamin D deficiency

Beyond peripheral mechanisms, vitamin D may influence central pain processing. Vitamin D receptors are expressed in neurons and glial cells within regions implicated in migraine pathophysiology, including the cortex, thalamus, hypothalamus, and brainstem nuclei. Hypovitaminosis D has been associated with increased pro-inflammatory cytokines, enhanced nitric oxide signaling, and modulation of CGRP-related pathways, all of which are central to trigeminovascular activation.^5,18 Deficiency may therefore create a pro-inflammatory and hyperexcitable neural environment, lowering the threshold for sustained nociceptive transmission and facilitating central sensitization.

A possible synergistic model

Pain in CM often differs from EM and may resemble tension-type headache, as acknowledged in ICHD-3.² Central sensitization is considered a dominant mechanism; however, peripheral muscular factors may contribute to the tension-type-like phenotype observed in CM. We propose a synergistic model in which vitamin D deficiency enhances peripheral musculoskeletal dysfunction (muscle fiber atrophy, trigger point formation, periosteal nociception), thereby causing musculoskeletal pain. Sustained peripheral input may then reinforce central sensitization, trigeminovascular activation, and headache chronification. In this framework, hypovitaminosis D acts not merely as a comorbidity but as a permissive or amplifying factor linking musculoskeletal dysfunction and persistent headache.

Potential explanation for vitamin D deficiency in migraine

Given the cross-sectional design, the directionality of the observed associations cannot be determined. It remains plausible that lower vitamin D levels are a consequence rather than a cause of increased migraine burden. Vitamin D deficiency is primarily caused by insufficient sun exposure, as sunlight is the main source for skin synthesis. Patients with CM may have reduced outdoor activity and physical mobility due to photophobia, leading to decreased sunlight exposure and vitamin D deficiency. In this context, hypovitaminosis D may represent an epiphenomenon of migraine burden rather than a causal contributor to chronification, indicating reverse causation. Thus, the observed association between vitamin D deficiency and migraine may reflect behavioral and lifestyle consequences of increased migraine burden rather than a direct pathophysiological effect.

Strengths and limitations

Strengths of this study include the three-arm design with EM and CM groups defined using standardized ICHD-3 diagnostic criteria, age- and sex-matched controls, comprehensive clinical assessment, and the use of both crude and adjusted statistical models. The inclusion of effect size measures enhances the interpretability of findings.

Several limitations should be acknowledged. First, the cross-sectional design precludes causal inference. Second, residual confounding factors such as dietary intake, sunlight exposure, and physical activity were not directly quantified. However, the primary objective of this study was not to determine the causes of vitamin D deficiency, but rather to examine the association between serum vitamin D levels and headache frequency and chronicity. Third, vitamin D was measured at a single time point, which may not fully reflect long-term status. Fourth, although statistically significant, the observed correlation between vitamin D and tenderness was moderate in magnitude. Fifth, the EM group may have been heterogeneous with respect to baseline headache frequency. Patients with high-frequency EM may clinically resemble those with CM, potentially influencing group comparisons. Stratification of EM into low-, moderate-, and high-frequency subgroups was not performed in this study and represents a limitation. Sixth, the high prevalence of vitamin D deficiency in the study population may have reduced the contrast between groups and could limit generalizability to populations with different baseline vitamin D status. Seventh, measures of migraine-related disability (e.g. HIT-6, MIDAS), severity of photophobia, and time spent outdoors were not assessed in this study. These factors may influence both migraine burden and vitamin D status and represent potential confounders. Future studies incorporating these variables may provide a clearer understanding of the relationship between vitamin D levels and migraine. Finally, this study was conducted in a single geographical region, and vitamin D status is influenced by latitude, climate, cultural practices, and ethnicity; therefore, the findings may not be generalizable to other populations.

Future studies should employ longitudinal designs with repeated assessments of headache burden, sunlight exposure, physical activity, and vitamin D status to better clarify temporal relationships. Randomized controlled trials of vitamin D supplementation in carefully phenotyped migraine populations may further help determine whether correction of hypovitaminosis D influences migraine progression or associated musculoskeletal tenderness.

Conclusion

In summary, serum vitamin D levels are significantly lower in CM than in EM and healthy controls, with a graded association across migraine categories. Vitamin D deficiency is independently associated with CM, and lower vitamin D levels correlate with increased muscle tenderness. These findings support a potential role of vitamin D in migraine pathophysiology and warrant further longitudinal and interventional studies to determine causality and therapeutic implications.

Clinical implications

Vitamin D deficiency may represent a modifiable factor in a subset of patients with chronic headache.

Screening for vitamin D deficiency may be particularly relevant in individuals with CM presenting with musculoskeletal pain, musculoskeletal tenderness, and fatigue.

Footnotes

ORCID iDs

Sanjay Prakash

Manju Yadav

Varoon Vadodaria

Sujal Mahendrabhai Patel

Ethical considerations

This study was approved by the university's Ethics Committee (SVIEC/ON/MED/S/RP/19020/24/11).

Consent to participate

Participants provided written informed consent before participation. Written informed consent was taken from the patients to publish the report.

Consent for publication

The authors give their consent for the publication of this manuscript in Cephalalgia Reports.

Author contributions

Sanjay Prakash: writing—original draft, writing—review & editing, methodology, validation, Supervision, funding acquisition, formal analysis, data curation, conceptualization, project administration, resources. Manju Yadav: writing—review & editing, formal analysis, data curation, software. Varoon Vadodaria: writing—review & editing, data curation, investigation, visualization. Sujal Patel: writing—review & editing, formal analysis, visualization.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

The data supporting the findings of this study is available from the corresponding author upon reasonable request.

Previous presentation

This manuscript was not presented at any meeting or published, nor is being considered for publication elsewhere.

AI usage declaration

The authors used Quillbot for technical editing and language refinement during the development of this manuscript. The final content was reviewed, verified, and approved by all authors. No AI tool was used for data collection, analysis, or the formulation of clinical conclusions.

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