Bruxism and Stress: Ultrasonographic Masseter Muscle Assessment and Salivary Melatonin‐Cortisol Dynamics

ABSTRACT

Background

Bruxism is a parafunctional activity characterised by teeth grinding or clenching, often associated with stress and neurophysiological factors. Its aetiology is multifactorial, with recent studies emphasising neurotransmitters and hormonal imbalances, particularly melatonin and cortisol. This study assesses masseter muscle thickness using ultrasonography and examines the relationship between salivary melatonin and cortisol levels in bruxism.

Objective

This study aimed to evaluate the ultrasonographic characteristics of the masseter muscle in individuals with bruxism and investigate the association between salivary melatonin and cortisol levels.

Methods

This cross‐sectional study included 80 participants (38 bruxists, 42 controls), aged 20–25 years. Bruxism was diagnosed through clinical examination and self‐reported questionnaires. Masseter muscle thickness was measured via ultrasonography in relaxed and contracted states. Salivary melatonin and cortisol levels were analysed using enzyme‐linked immunosorbent assay (ELISA). Group comparisons were conducted using independent t‐tests and ANOVA, with correlation analyses using Pearson's tests.

Results

Salivary melatonin levels showed no significant difference between groups (p = 0.067), while salivary cortisol levels were significantly higher in the bruxism group (p = 0.001). No significant differences in masseter muscle thickness were observed in either state (p > 0.05). A weak but significant positive correlation existed between melatonin and cortisol levels (p < 0.05).

Conclusion

Although not statistically significant, melatonin levels tended to be higher in bruxist patients, possibly due to a negative feedback mechanism to prevent the side effects of cortisol such as oxidative stress or a balancing process of these hormones by cytokines involved such as IL‐1β.

1. Introduction

Bruxism is defined as an oral movement disorder characterised by teeth clenching or grinding. This condition can occur both during sleep and wakefulness, with an estimated prevalence of approximately 8%–10% in adults [1]. Bruxism has a multifactorial aetiology. Previously, occlusal discrepancies and craniofacial anatomical anomalies were considered the primary causes of bruxism. However, recent studies suggest that these factors have minimal influence on its development. Moreover, bruxism has been demonstrated to be part of the transition between sleep and wake cycles. Additionally, various neurotransmitters have been implicated in the pathophysiology of bruxism. Other contributing factors include smoking, systemic diseases, trauma, genetic predisposition and the use of substances such as alcohol, caffeine, recreational drugs, and certain medications (e.g., L‐dopa, SSRIs, propranolol), which are believed to play a role in its pathogenesis [2, 3, 4, 5]. Although extensive research has been conducted on the prevalence, causes, effects and management of bruxism, no established consensus or definitive guideline currently exists for clinical dental practice. Bruxism is known to have detrimental effects on oral tissues and the masticatory system. However, diagnosing bruxism and understanding its functional implications in clinical practice remain challenging [6]. Some studies suggest that modifications in occlusal contacts, such as increasing the vertical dimension, can temporarily reduce bruxism but are not effective in eliminating it entirely [7, 8, 9].

Various methods have been employed to assess bruxism activity. Among these, self‐reported questionnaires are the most widely used. Additionally, clinical examination and the observation of dental wear patterns are frequently utilised in both clinical and research settings. Some studies have also implemented intraoral devices to detect bruxism activity. These studies involve evaluating wear on occlusal splints, measuring the forces applied to these devices and detecting tooth contacts [10, 11].

The most recent and widely accepted approach for defining and assessing bruxism in the literature is the Standardised Tool for the Assessment of Bruxism (STAB), developed by Manfredini et al. This tool aims to provide a comprehensive and consistent evaluation framework for both clinical and research applications [12].

A review of the literature suggests that melatonin may play a role in the treatment of bruxism [13]. Given the therapeutic benefits observed in melatonin replacement therapy, melatonin deficiency has been proposed as a potential etiological factor for bruxism [14]. Furthermore, these findings highlight the importance of investigating the role of oxidative stress‐related biomarkers, hormones, or proteins with antioxidant properties in the pathophysiology of bruxism through salivary or blood samples. This study also examines salivary cortisol levels, as cortisol is secreted by the adrenal cortex in response to hypothalamic–pituitary–adrenal (HPA) axis activation and is involved in regulating pro‐inflammatory processes. Salivary cortisol levels are considered a potential biomarker for stress and depression [15].

The primary aim of this study is to evaluate salivary cortisol, melatonin levels, and also masseter muscle thickness in clinically diagnosed individuals with bruxism using ultrasonography (USG). Previous studies have shown that bruxism leads to an increase in the thickness of the masseter and temporalis muscles due to muscular adaptation. However, it remains unclear whether these muscular changes parallel alterations in hormonal balance. Therefore, this study also aims to assess changes in salivary melatonin and cortisol levels in individuals with bruxism and to investigate whether there is a relationship between muscle adaptation and hormonal fluctuations.

This study is based on two null hypotheses:

There is no difference in masseter muscle thickness between individuals with and without bruxism.

There is no difference in salivary melatonin and cortisol levels between individuals with and without bruxism.

2. Methods

2.1. Study Design and Ethical Approval

This cross‐sectional study was conducted after obtaining ethical approval from the Clinical Research Ethics Committee of Van Yuzuncu Yil University (Approval No: 2024/10‐31). Written informed consent was obtained from all participants before inclusion in the study.

2.2. Power Analysis and Sample Size Determination

The sample size was determined via a priori power analysis conducted using G*Power software (version 3.1) to ensure sufficient statistical power for detecting potential group differences in salivary cortisol and melatonin levels. For cortisol, data reported by Miletić et al. (2018) were used, in which mean salivary cortisol levels were 45.75 ± 17.54 nmol/L in the bruxism group and 34.42 ± 7.80 nmol/L in the control group. Based on these values, the calculated effect size was Cohen's d = 0.93. Assuming a significance level of α = 0.05 and a power of 1 − β = 0.80, the minimum required sample size was calculated to be 19 participants per group [16]. Based on data from a previous study by Süzen and Delilbaş (2023), the following parameters were used: effect size = 0.542, standard deviation = 0.73, significance level (α) = 0.05, and power (1 – β) = 0.80. This calculation showed that a minimum of 11 participants per group was required [17]. This study included 40 participants in each group, which exceeds these thresholds and ensures adequate power to detect differences in both biomarkers.

2.3. Participants and Study Groups

The study included 80 participants aged 20–25 years, consisting of 38 bruxists and 42 non‐bruxist controls. All participants were fourth‐ and fifth‐year dentistry students at our faculty. This specific age group and educational background were chosen to standardise saliva collection procedures and minimise variability in stress levels and lifestyle factors. To ensure balanced group comparisons, gender distribution was equalised. Bruxism group: 19 females (50%) and 19 males (50%). Non‐bruxist control group: 21 females (50%) and 21 males (50%).

2.4. Inclusion and Exclusion Criteria

Inclusion criteria for the bruxism group: Clinically observed occlusal/incisal tooth wear and fractures. Presence of pronounced linea alba.

2.5. Positive Findings in the Self‐Reported Bruxism Questionnaire

Bruxism history within the last 2 years based on clinical and radiological examination. Exclusion criteria for both groups: Individuals not consenting to ultrasonographic or clinical examination. Missing molars, acute infections, or recent orthodontic treatment. Use of bruxism appliances or botulinum toxin injections in the masseter muscle within the last 6 months. Use of antidepressants, sleep disorder medications, or melatonin supplements. Presence of systemic diseases, endocrine disorders, or genetic syndromes. Diagnosis of temporomandibular disorder (TMD) based on joint sounds, limited jaw movements, or pain on palpation. History of psychiatric treatment or medication use. Body mass index (BMI) outside the WHO‐defined range for the age group.

2.6. Self‐Reported Bruxism Questionnaire and Clinical Examination

Bruxism symptoms were assessed using the Self‐Reported Bruxism Questionnaire (SBQ), a validated tool that categorises participants based on symptom severity [18].

1. ‘Do you notice yourself grinding or clenching your teeth while sleeping?’

2. Observer‐Reported Grinding. ‘Has anyone observed or mentioned that you grind your teeth during sleep?’

3. Tooth Wear Observation:‘Have you noticed excessive wear or flattening of your teeth that seems abnormal?’

4. ‘Do you wake up with fatigue, tightness, or tenderness in your jaw muscles?’

5. ‘Upon waking, do you feel like your teeth were tightly clenched or that your mouth is sore?’

6. ‘Do you experience aching or pain in your temple area when you wake up?’

7. ‘Do you have difficulty opening your mouth fully in the morning?’

8. ‘Does your jaw joint feel stiff or tense upon awakening, requiring movement to relieve it?’

9. ‘Do you hear or feel a clicking/popping sound in your jaw joint when you wake up that later subsides?’

10. ‘If you were to rate your pain on a scale from 0 (no pain) to 10 (worst possible pain), what number would you choose?’

Participants scoring 1–10 were classified as unlikely to have bruxism, while those scoring 10–28 were classified as highly likely to have bruxism. Clinical examination was performed by two experienced radiologists (12 years of experience in dentistry). Assessments included intraoral and extraoral examination, with specific evaluation of linea alba, occlusal/incisal tooth wear and fractures.

2.7. Ultrasonographic Assessment of Masseter Muscle Thickness

Masseter muscle thickness was measured using USG (Mindray DC‐60, Nanshan, Shenzhen, China). A 14–16 MHz linear transducer (L46NE × 6 cm depth) was used. The skin surface was cleaned with alcohol, and ultrasound gel (Naturel Ultrason Gel, Turkey) was applied for optimal acoustic transmission. Participants were seated in a neutral, upright position, looking straight ahead to avoid external pressure on the masseter muscle.

2.8. Measurement Protocol

Each participant underwent six measurements per side to ensure reliability. Measurements were taken in two positions: relaxed state (muscle at rest). Contracted state maximum voluntary clenching (Figures 1 and 2). The transducer was placed perpendicular to the muscle fibres to obtain real‐time images. To enhance measurement consistency, evaluations were performed by two independent radiologists with 6–10 years of experience in radiological assessments.

FIGURE 1.

The ultrasonographic image of the masseter muscle at rest.

FIGURE 2.

The ultrasonographic image of the masseter muscle during contraction.

2.9. Sample Collection and Laboratory Analysis

Saliva samples were collected at a standardised time, between 08:00–08:30 AM, approximately 30 min after participants woke up, to ensure the highest hormonal levels were captured. To prevent variability in hormone levels, participants were required to fast and brush their teeth with a toothbrush only (without toothpaste or mouthwash) at least 30 min before sampling [19]. Saliva samples were collected in the clinic under standardised conditions. To obtain stimulated saliva, participants chewed paraffin gum before collection. A total of 3 mL of saliva was collected from each participant. Immediately after collection, the samples were stored at −45°C to preserve hormonal stability until analysis. Collected saliva samples were stored in Eppendorf tubes until analysis. Salivary melatonin and cortisol levels were measured using commercial ELISA kits (Sunlong Biotech, China), following the manufacturer's instructions. Salivary melatonin levels were expressed in pg/mL and salivary cortisol levels in ng/mL. The cortisol and melatonin levels were measured once each in the samples. The measurement range of the melatonin kit was 3–180 pg/mL, Sensitivity: 0.5 pg/mL, Intra assay: CV < 10%, Inter assay: CV < 12%. The measurement range of the cortisol kit was 3–100 ng/mL, Sensitivity: 0.5 ng/mL, Intra assay: CV < 10%, Inter assay: CV < 12%.

2.10. Statistical Analysis

Statistical analyses were conducted using SPSS software (version 2020). The Shapiro–Wilk test was used to assess the normality of the data, while the Levene's test evaluated variance homogeneity. As the data were normally distributed, descriptive statistics were presented as mean and standard error. Comparisons between the bruxism and control groups for salivary melatonin and cortisol levels, as well as masseter muscle thickness in relaxed and contracted states, were performed using one‐way ANOVA followed by an independent t‐test. Pearson's correlation analysis was used to evaluate the relationships between salivary cortisol levels and masseter muscle thickness in both contracted and relaxed states.

3. Results

The mean age of the participants was calculated as 23.05 years. There was no significant difference in the mean ages between the control and bruxism groups (p > 0.05). Additionally, the salivary melatonin levels of the control and bruxism groups were similar, with no statistically significant difference between them (p = 0.067). However, the salivary cortisol levels in the bruxism group were significantly higher than those in the control group (p = 0.001, Table 1, Figure 3).

TABLE 1.

The mean and standard deviation values of melatonin and cortisol levels were measured in saliva samples.

Group	Melatonin (pg/mL)	Cortisol (ng/mL)
	Mean ± SD	Mean ± SD
Control	11.38 ± 1.72	14.82 ± 1.67
Bruxism	12.06 ± 1.72	16.77 ± 1.63
p *	0.067	0.001

Abbreviation: SD, standard deviation.

^*Independent samples t‐test, p < 0.05.

FIGURE 3.

Comparison of melatonin and cortisol levels measured in saliva samples, *p < 0.05.

No statistically significant difference was observed in masseter muscle thickness between bruxist and non‐bruxist individuals in either the contracted or relaxed state (Table 2). However, when comparing masseter muscle thickness between genders, a statistically significant difference was found in all positions except for the right relaxed position (Table 3).

TABLE 2.

Statistical evaluation of masseter muscle thickness in relaxed and contracted conditions between genders.

Masseter thickness	Gender	N	Mean ± SD	p *
Relaxed‐right	Female	38	1.00 ± 0.18	0.139
	Male	42	1.05 ± 0.17
Contracted‐right	Female	38	1.33 ± 0.22	< 0.001
	Male	42	1.51 ± 0.26
Relaxed‐left	Female	38	1.00 ± 0.16	0.007
	Male	42	1.10 ± 0.18
Contracted‐left	Female	38	1.37 ± 0.22	0.007
	Male	42	1.56 ± 0.26
Mean relaxed	Female	38	1.35 ± 0.20	< 0.001
	Male	42	1.54 ± 0.25
Mean contracted	Female	38	1.00 ± 0.16	0.025
	Male	42	1.07 ± 0.16

Note: N: sample size.

Abbreviation: SD, standard deviation.

^*Independent samples t‐test, Bold values indicate statistically significant differences, p < 0.05.

TABLE 3.

Statistical evaluation of masseter muscle thickness in relaxed and contracted positions in bruxist and non‐bruxist individuals.

Masseter thickness	Bruxism condition	N	Mean ± SD	p *
Relaxed‐right	Bruxist	38	1.03 ± 0.18	0.92
	Non‐bruxist	42	1.03 ± 0.17
Contracted‐right	Bruxist	38	1.47 ± 0.26	0.23
	Non‐bruxist	42	1.40 ± 0.26
Relaxed‐left	Bruxist	38	1.05 ± 0.17	0.79
	Non‐bruxist	42	1.06 ± 0.17
Contracted‐left	Bruxist	38	1.50 ± 0.28	0.36
	Non‐bruxist	42	1.45 ± 0.24
Mean relaxed	Bruxist	38	1.49 ± 0.26	0.27
	Non‐bruxist	42	1.43 ± 0.24
Mean contracted	Bruxist	38	1.04 ± 0.16	0.93
	Non‐bruxist	42	1.04 ± 0.17

Note: N: sample size.

Abbreviation: SD, standard deviation.

^*Independent samples t‐test, p < 0.05.

Additionally, a statistically significant weak positive correlation was observed between salivary melatonin and cortisol levels (rs = 0.288, n = 80; 38 bruxist and 42 non‐bruxist participants, p = 0.007). In order to evaluate the relationship between salivary cortisol levels and masseter muscle thickness, Pearson's correlation analysis showed weak and statistically non‐significant negative correlations between salivary cortisol levels and masseter muscle thickness in both states: Contracted: r = −0.072, p = 0.512, Relaxed: r = −0.153, p = 0.164.

To assess the inter‐rater reliability of ultrasonographic measurements, the inter‐class correlation coefficient (ICC) was used. The analysis revealed a high ICC value of 0.918 for masseter muscle thickness, indicating strong agreement between the measurements made by both examiners. This high ICC value demonstrates that the measurements are highly reliable and yield reproducible results.

Additionally, one of the researchers performed measurements on 16 patients, which represents 20% of the total sample size, further supporting the consistency of the findings. The strong inter‐rater reliability values confirm the reliability and reproducibility of the measurements taken by different examiners during the study. These findings emphasise the robustness of the measurement methodology and ensure the reliability of the data collected.

4. Discussion

Reis Durão et al. [20] conducted a systematic review on the diagnosis of bruxism and highlighted several advantages of USG, including its ability to clearly visualise superficial muscles such as the masseter and temporalis, its radiation‐free nature, simplicity, cost‐effectiveness, portability and lack of biological side effects. These features suggest that USG may be preferred over other imaging methods such as magnetic resonance imaging (MRI) and computed tomography (CT) in the clinical evaluation of bruxism patients [21, 22, 23].

Serra‐Negra et al. (2013) suggested that poor sleep quality significantly impacts individuals with nocturnal or daytime bruxism [24]. Gürhan et al. (2024) investigated melatonin hormone levels in bruxist and non‐bruxist individuals and found that melatonin levels were lower in the bruxist group compared to the control group [25]. Similarly, Gaio et al. (2022) suggested that deficiencies in melatonin hormone or melatonin receptors may contribute to sleep bruxism and that this condition may vary across different ethnic groups [26]. Erden (2019) reported a significant reduction in bruxism symptoms in children following melatonin treatment but noted that these studies were insufficient to reach a definitive conclusion and had certain limitations [27]. A study examining the cortisol‐melatonin equilibrium in the literature highlights that norepinephrine, the primary neurotransmitter of the pineal gland, regulates the ratios of melatonin and corticotropin‐releasing hormone (CRH). CRH, in turn, has been shown to elevate cortisol concentrations. Activated macrophages, microglia, and their osseous variant, osteoclasts, possess the capacity to secrete interleukin‐1β (IL‐1β). IL‐1β may upregulate norepinephrine, subsequently increasing cortisol via CRH while concurrently suppressing melatonin levels. This suggests the existence of a negative feedback mechanism between melatonin and cortisol [28]. This current study, which measured the inverse relationship between melatonin and cortisol in bruxers versus healthy controls, aligns with these findings.

Salameh et al. (2014), Suprajith et al. (2022) and Alsahman et al. (2024) evaluated salivary cortisol levels in individuals with temporomandibular joint (TMJ) disorders and concluded that cortisol levels were higher in individuals with TMJ dysfunction [29, 30, 31]. Similarly, Fluerasu et al. (2021) found that salivary cortisol levels were higher in both bruxist individuals and females [14]. Glucocorticoids in the HPA axis function as potent anti‐inflammatory agents in the body, primarily mediating their effects through monocyte and macrophage apoptosis. While this inflammatory regulation occurs effectively under basal conditions, chronic stress states can trigger distinct mechanistic pathways, potentially leading to HPA axis dysregulation and associated increases in inflammation. This adaptive process, termed ‘glucocorticoid resistance’, is characterised by reduced sensitivity of immune cells to glucocorticoid signalling [32, 33]. To minimise potential confounding effects of cortisol resistance or HPA axis dysregulation and ensure sample homogeneity, our study specifically recruited university students within a narrow age range and comparable educational backgrounds.

Sriharsha et al. (2018) also assessed changes in cortisol hormone levels after soft occlusal splint therapy in bruxist individuals. They found that although cortisol levels decreased after treatment, this reduction was not significant [34]. Additionally, Castano et al. reported that melatonin supplementation in fibromyalgia patients resulted in reduced anxiety and cortisol levels [35]. Several studies have demonstrated a relationship between melatonin and cortisol. Research has shown that acute stress triggers a biological adaptation response in which cerebral serotonin (a precursor of melatonin) availability increases, promoting a negative feedback mechanism in the hypothalamic–pituitary–adrenal (HPA) axis to regulate cortisol levels [35, 36]. However, chronic stress leads to a reduction in serotonin levels in the synaptic cleft and stimulates neuronal reuptake, potentially causing HPA‐axis hyperactivity and the onset of stress‐related affective disorders [37]. A recent rodent study demonstrated that melatonin alleviates chronic stress‐induced hippocampal microglial pyroptosis and subsequent depression‐like behaviours in rats by inhibiting the Cathepsin B/NLRP3 signalling pathway [38]. In this study, the observed slight increase in melatonin levels despite significant cortisol elevation may suggest the involvement of distinct independent regulatory pathways between these hormones.

In this study, salivary cortisol levels were markedly higher in the bruxism group, whereas melatonin levels were slightly elevated. This finding suggests that the body's endocrine system may have physiologically compensated by increasing endogenous melatonin secretion to counterbalance the elevated cortisol levels observed in individuals with bruxism. This trend may indicate a compensatory or feedback mechanism; however, due to the weak correlation, causal inference cannot be made based on the current cross‐sectional data. An important contribution of our study is the combined evaluation of salivary melatonin and cortisol levels alongside ultrasonographic assessment of masseter muscle thickness in individuals with bruxism anintegrative approach that is not commonly found in the existing literature.

No statistically significant differences in masseter muscle thickness were observed between individuals with and without bruxism. Several factors may help explain this finding. Although participants with a history of bruxism within the past 2 years were included, the precise duration and frequency of the habit could not be objectively determined. Since muscle adaptation is influenced by both the intensity and chronicity of parafunctional activity, such variability may have affected the outcomes. Moreover, the SBQ questionnaire used in this study assessed bruxism collectively, without distinguishing between sleep and awake subtypes. Given that these subtypes may involve different patterns of muscle activity, the lack of differentiation might have contributed to the non‐significant results. Additionally, interindividual variability in physiological muscle responses, as well as potential compensatory activity by other masticatory muscles, may have masked subtle changes in the masseter.

While cortisol is a widely recognised biomarker of stress, its influence on muscle morphology, particularly in the context of bruxism, may be indirect or modulated by additional factors such as sleep quality, clenching intensity, or the duration of parafunctional activity. These findings indicate that salivary cortisol levels are not significantly associated with masseter muscle thickness, suggesting that other physiological or behavioural mechanisms may underlie the variations observed. Further research incorporating a broader range of stress‐related biomarkers and objective stress assessment tools is warranted to better elucidate this complex interaction.

4.1. Limitations

This study has some limitations that should be considered when interpreting the results. Although saliva collection procedures were carefully standardised, individual variations in circadian rhythms and daily stress levels might have influenced hormonal measurements. The study population consisted of a relatively homogeneous group of dental students aged 20–25 years, which may slightly limit the generalisability of the findings to the wider population. Additionally, bruxism diagnosis was based on a self‐reported questionnaire supported by clinical examination, without the use of objective diagnostic tools such as polysomnography or electromyography. Although confounding variables such as gender and BMI were controlled through matching and exclusion criteria, other influential factors such as recent stress exposure, sleep quality and chronotype were not quantitatively assessed. Therefore, it remains unclear whether the observed hormonal trends would persist after statistically controlling for these unmeasured confounders. Future studies should employ multivariate modelling approaches to account for these variables.

5. Conclusion

In our study, salivary cortisol levels were significantly higher in the bruxism group than in the control group, suggesting a heightened stress response in individuals with bruxism. Melatonin levels were slightly higher in bruxism patients. This trend may reflect a potential compensatory response to cortisol‐mediated oxidative stress or a cytokine‐regulated feedback mechanism. While this hypothesis is biologically plausible, the weak correlation and cross‐sectional design of the study preclude definitive causal interpretations. Future longitudinal studies incorporating additional physiological parameters may provide deeper insights into these associations. The weak correlation between cortisol and melatonin, albeit significant, indicates that other factors are more important for cortisol levels than melatonin levels and vice versa.

Author Contributions

S.K. and A.K. contributed to the conceptualisation and supervision of the study. S.K., A.K. and Z.H. were involved in the methodology, software, validation, formal analysis, investigation, resources, data curation, original draft preparation and review and editing of the manuscript. S.K. was responsible for the visualisation. All authors have read and agreed to the published version of the manuscript.

Ethics Statement

This cross‐sectional study adhered to the principles outlined in the Declaration of Helsinki. All procedures performed in this study involving human participants were in accordance with and the study has been approved by the ethical standards of the institutional local committee of Van Yuzuncu Yil University and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. (Approval No: 2024/10‐31).

Consent

Informed consent was obtained from all participants.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The datasets used and analysed during the current study are available from the corresponding author upon reasonable request.