Salivary Melatonin Levels in Comparison with Disease Features and Antihistamine Use: Prospective Follow-Up of Atopic Dermatitis Patients

1. Introduction

Atopic dermatitis (AD) is a common chronic inflammatory skin disease affecting both children and adults. The main symptom, pruritus, is frequently associated with sleep disturbances, and a vicious itch–scratch cycle where skin inflammation and disease severity worsen as the patient scratches for relief [1,2,3,4]. Persistent pruritus has a profound negative impact on patients' quality of life, provoking repetitive scratching that compromises skin barrier integrity, promotes cutaneous inflammation and increases the risk of secondary infection. In addition, chronic pruritus is associated with adverse psychosocial outcomes, including impaired concentration, anxiety, and depression [5]. Also, sleep disturbance, as a major clinical AD manifestation, is noted as an important component of disease burden and is incorporated into AD severity assessment using the Scoring Atopic Dermatitis (SCORAD) index [6]. The prevalence of sleep disturbances among AD patients has been estimated at 50–80% in children and 30–90% in adults [7,8]. Sleep abnormalities may occur throughout all stages of sleep and include prolonged sleep onset latency, frequent nocturnal waking, difficulty waking up, and excessive daytime sleepiness. However, adequate sleep is essential for maintaining overall health, since both acute and chronic sleep disturbances are associated with a wide range of cognitive, behavioral, and mood impairments. These disturbances have been linked to poorer academic performance and impose a substantial socioeconomic burden on patients, their families, and society through increased healthcare utilization, higher medical and treatment-related costs, and reduced productivity [9,10].

Melatonin (N-acetyl-5-methoxytryptamine) is a molecule that plays a key role in sleep disturbances. It is primarily synthesized in the pineal gland from the amino acid tryptophan and is subsequently secreted into the bloodstream, cerebrospinal fluid, and various peripheral tissues, including the retina, bone marrow, gonads, gastrointestinal mucosa, and the skin [11,12,13]. Melatonin secretion follows a circadian rhythm, with nocturnal concentrations approximately tenfold higher than daytime levels. Exposure to light suppresses melatonin secretion, thereby promoting wakefulness, whereas the absence of light during the night increases melatonin production, inducing sleepiness and facilitating sleep onset. Melatonin exerts its biological effects through G protein-coupled membrane receptors expressed in the brain and peripheral organs, as well as through nuclear receptors and calmodulin. Membrane melatonin receptors include two major subtypes: the high-affinity Mel1a receptors (ML1, ML1a, MT1, and MTNR1A), predominantly distributed in the brain and skin, and the low-affinity Mel1b receptors (MT2, ML1b, and MTNR1B), which are distributed throughout various regions of the brain [14,15]. Melatonin regulates sleep by acting on the suprachiasmatic nucleus, the principal circadian pacemaker [16]. Higher serum melatonin levels have been associated with improved sleep quality, reduced sleep fragmentation, and decreased disease severity in conditions such as AD.

Among its various roles, including maintaining skin barrier homeostasis, melatonin is a potent antioxidant. In keratinocytes and mitochondria, melatonin scavenges reactive oxygen and nitrogen species, enhances the activity of antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase, and increases intracellular glutathione levels [16,17]. During chronic inflammation, characterized by elevated inflammatory mediator levels and reactive free radicals, melatonin suppresses pro-inflammatory pathways by reducing heat shock protein 70 (Hsp70) expression and inhibiting IL-1β, caspase-1, and caspase-3 activity in keratinocytes. It also activates antioxidant enzymes, reduces reactive oxygen species production, inhibits nuclear factor kappa B (NF-κB) translocation, and suppresses 5-lipoxygenase activity, thereby limiting cytokine production and leukocyte migration [16,17]. In addition, melatonin exerts immunomodulatory effects by enhancing both innate and adaptive immune responses through increased cytokine secretion and neutrophil chemotaxis. It upregulates the expression of immune markers, including CD69, CD28, and BCL2, resulting in increased CD4+ T-cell production. Furthermore, increased interleukin-4 (IL-4) promotes a Th2 immune response, whereas reduced interferon-gamma (IFN-γ) expression suppresses the Th1 response [14,16]. The skin represents a major extrapineal source of melatonin and its metabolites, and studies have demonstrated that melatonin concentrations are significantly higher in the skin than in serum, supporting its autocrine and paracrine activity. Furthermore, the autonomous cutaneous melatonin system comprises various skin cells, including keratinocytes, melanocytes, and fibroblasts, and contains the enzymes required for melatonin biosynthesis, including tryptophan hydroxylase, arylalkylamine N-acetyltransferase, and hydroxyindole-O-methyltransferase [18]. For research purposes, melatonin concentrations can be determined in blood, urine, or saliva [19]. In saliva, melatonin is present exclusively in its free, albumin-unbound form [20]. When measuring melatonin concentrations, both the timing and the sampling method should be carefully considered [14]. Previous studies have shown that salivary and plasma melatonin concentrations may differ by as much as 36%. Salivary melatonin concentrations also exhibit marked circadian variation, typically ranging from 1 to 5 pg/mL during the daytime and from 10 to 50 pg/mL during the night [21,22].

Pruritus in AD is commonly managed with a combination of preventive measures, symptomatic therapies, including emollients and topical or systemic corticosteroids, and immunomodulatory agents. Oral antihistamines (AHs) (H1-AHs) are also frequently prescribed despite limited and conflicting evidence of their clinical efficacy. Although current clinical guidelines for the management of AD do not mention the use of AHs, short-term use of 1st-generation AHs has been mentioned as useful therapy option for the management of pruritus-related insomnia in AD patients [5,23,24]. A systematic review of 16 studies reported inconclusive evidence supporting the use of AHs in AD patients, although the available data suggest that sedating AHs may improve sleep quality [5,25]. In contrast, the use of 2nd-generation non-sedating AHs is generally not recommended for the management of AD, except in patients with concomitant urticaria or atopy. Although early randomized, double-blind, crossover trials concluded that AHs were ineffective in relieving pruritus associated with AD, the Early Treatment of the Atopic Child (ETAC) trial demonstrated a trend toward less severe pruritus in infants treated with cetirizine compared with placebo; however, this difference did not reach statistical significance [5,26].

Therefore, the aim of the present study was to evaluate melatonin levels in AD patients in relation to the clinical characteristics of the disease and the use of oral H1-AHs (no AH use, introduction of AH therapy, or discontinuation of oral AH therapy) during a prospective follow-up.

2. Results

Data on clinical scores and melatonin levels on study participants with descriptive statistics at initial measurement (T1) are presented in Table 1.

2.1. Association Between Salivary Melatonin Levels and Clinical Scores for Atopic Dermatitis

2.1.1. Disease Severity (SCORAD, EASI) and Pruritus Intensity (Peak Pruritus Numeric Rating Scale/NRS) in Relation to Salivary Melatonin Levels, Assessed at the First Sampling T1

At the first sampling point, EASI/disease severity showed a statistically significant negative linear correlation with salivary melatonin levels (r=-0.344; p=0.009; Table 2). Melatonin levels were not significantly correlated with either SCORAD or NRS; however, the correlation with NRS approached statistical significance (r=−0.248, p=0.063).

2.1.2. Comparison Between Disease Severity Measures (SCORAD, EASI), Pruritus Intensity (NRS), and Sleep Quality (PSQI), Assessed at the Second Sampling T2

At the second sampling point, SCORAD correlated with EASI (r=0.843; p<0.001), NRS (r=0.304; p=0.022), and the Pittsburgh Sleep Quality Index (PSQI) (r=0.491; p<0.001; Table 3). EASI correlated with NRS (r=0.381; p=0.003) and PSQI (r=0.453; p<0.001) (Table 3).

2.3. Changes in Clinical and Salivary Melatonin Parameters During Prospective Follow-Up in Relation to Antihistamine Use

2.3.1. Comparison of the Initial (T1) and Second Measurements (T2) After Discontinuation of Antihistamines, in Relation to Changes in Disease Severity Indicators (SCORAD, EASI) and Salivary Melatonin Levels

In patients where AHs were discontinued (N=8), a significant increase in SCORAD was observed (p=0.012; r=0.892), as well as EASI (p=0.012; r=0.893), and in NRS (p=0.010; r=0.907); however, no significant change was found in melatonin levels (Figure 1).

2.3.2. Comparison of the First and Second Measurements After Initiation of Antihistamine Therapy, with Respect to Changes in Disease Severity (SCORAD, EASI) and Salivary Melatonin Levels

In those for whom AHs were introduced (N=16), a significant decrease was observed in SCORAD (p=0.012; r=0.889), EASI (p=0.012; r=0.879), and NRS (p=0.010; r=0.849), whereas no significant change was found in melatonin levels.

In the non-AH group (N=33), a significant decrease was observed in SCORAD (p=0.001; r=0.605) and EASI (p=0.001; r=0.590), but not in NRS or melatonin levels.

Sleep quality did not differ significantly between the three groups at T2.

When comparing the changes between the timepoints (ΔT1–T2), significant differences between groups were observed for the AD severity indicators, SCORAD, EASI, and NRS (p<0.001; η2=0.442-0.492), but not for melatonin (Table 4, Figure 2). Statistically significant differences were found between all three groups. Compared to the non-AH group, the group in which AHs were discontinued showed increased severity (SCORAD, EASI, NRS), whereas the group in which AHs were introduced showed an decrease in severity, i.e., SCORAD, EASI, and NRS scores.

Compared with the non-AH group, discontinuation of AHs was associated with a worsening AD clinical condition, reflected by increased symptom severity, and initiation of AH treatment was associated with an improved clinical condition, reflected in a reduction in symptoms.

3. Discussion

Since this pruritus impairs sleep quality of AD patients, melatonin is considered an important molecule in the pathophysiology of AD [27,28]. According to a study by Chang et al. disturbed sleep patterns are common in children with AD, affecting up to 60% of patients, and have a substantial negative impact on quality of life [29]. Among dermatological diseases, melatonin concentrations have been investigated most extensively in AD, but studies have predominantly been conducted in pediatric AD patients. In our study conducted on adults with AD, AD severity (index EASI) significantly correlated with salivary melatonin levels negatively and linearly, which supports an association between melatonin levels and the clinical AD characteristics.

The first study evaluating melatonin levels in AD was conducted by Schwarz et al. in 1988, who assessed serum melatonin levels in adult patients with severe AD [30]. Measurements were obtained every two hours over a 24-hour period, which showed predominantly reduced melatonin levels and an altered nocturnal melatonin secretion pattern, suggesting there is a disruption of circadian melatonin regulation in severe AD [30]. In other studies, an association between lower melatonin levels and greater AD disease has been consistently demonstrated. Jaworek et al. reported significantly lower serum melatonin concentrations in patients with very severe AD compared to those with less severe AD [11]. Similarly, Dwiyana et al. found significantly reduced serum melatonin levels in children with moderate AD compared with healthy controls [31]. In addition to serum measurements, melatonin has also been assessed in saliva. Kimata demonstrated significantly lower nocturnal salivary melatonin concentrations in 40 children with mild-to-moderate AD than in healthy controls (26.5 pg/mL vs. 58.6 pg/mL) [32].

Melatonin has been increasingly recognized for its potential role in mitigating AD and other allergic inflammatory diseases through multiple biological mechanisms. Cutaneous melatonin synthesis is regulated by the circadian rhythm and is upregulated in response to oxidative and environmental stress, thereby supporting the skin's adaptation to fluctuations in the light–dark cycle [18]. The potential role of melatonin in AD is further supported by its well-established anti-inflammatory and immunomodulatory properties. Melatonin contributes to skin barrier integrity preservation by limiting the chronic activation of inflammatory pathways. In particular, it inhibits activation of the transcription factor NF-κB, thereby reducing the expression of pro-inflammatory cytokines, including IL-1β and IL-6, as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)[18]. Furthermore, melatonin attenuates caspase activation, thereby limiting stress-induced apoptosis and inflammation and protecting against skin barrier dysfunction associated with chronic inflammation and oxidative stress. Melatonin also exerts broad immunomodulatory effects by influencing both innate and adaptive immune responses. It regulates T-cell differentiation and activation, promotes the production of IFN-γ and IL-2, and contributes to the maintenance of the balance among Th1, Th2, and Th17 immune responses. In addition, melatonin enhances the activity of regulatory T cells (Tregs), thereby promoting immune tolerance and preventing excessive inflammatory responses [15,18,33]. Melatonin modulates the secretion of both pro-inflammatory and anti-inflammatory cytokines and influences the proliferation and differentiation of immune cells [14]. Experimental studies further suggest that melatonin may ameliorate AD-associated inflammation by reducing total serum IgE and IL-4 levels [34]. Considering the close functional relationship between the cutaneous immune system and the epidermal barrier, melatonin immunomodulatory effects may further contribute to the maintenance of barrier integrity by limiting uncontrolled inflammatory activation and preventing tissue damage [18]. In addition, the skin exhibits circadian fluctuations in proliferative, reparative, and immune functions that are regulated by peripheral biological clocks, including clock genes such as PER1. Melatonin synchronizes these local circadian oscillators, thereby supporting epidermal homeostasis and tissue repair [18]. Emerging evidence also suggests that melatonin may influence the skin microbiome by modulating microbial composition, metabolite production, and epithelial barrier integrity, giving rise to the concept of the melatonin–circadian rhythm–skin microbiome axis. Collectively, the melatonin' antioxidant, anti-inflammatory, immunomodulatory, and anti-apoptotic properties, together with its potential effects on skin tight junction proteins and circadian regulation, support its role as a promising mediator of skin barrier homeostasis at both the cellular and molecular levels.

While previous clinical studies on melatonin and AD primarily focused on children, multiple significant human studies have specifically analyzed melatonin oral supplementation in adult patients. According to a recent systematic review with meta-analysis comparing melatonin supplementation (3–6 mg/day) with placebos, melatonin significantly reduced sleep-onset latency and improved clinical disease severity, while maintaining a favorable safety profile [1]. (Melatonin primarily facilitates sleep initiation rather than increasing total sleep duration) [1,35,36]. A significant improvement in SCORAD has also been reported, when comparing supplementation with placebos—following 6 weeks of melatonin supplementation in one study, SCORAD scores and serum total IgE levels improved, though without significant impact on pruritus scores, high-sensitivity C-reactive protein, sleep-onset latency, or total sleep time [37]. According to studies on children with AD, oral melatonin reduced sleep-onset latency by 21.4 minutes (compared to placebos) and decreased AD severity, assessed by SCORAD, without adverse effects [11,31,38,39].

As melatonin may improve AD condition, it may represent a promising therapeutic option for preserving skin integrity and maintaining epidermal barrier function [14]. Systemic melatonin may be considered as an adjunctive therapy for select patients, but routine use cannot be recommended currently due to limited clinical evidence. Thus, larger, well-designed randomized controlled trials are needed to establish its efficacy and safety [1,11,31,40]. In contrast, H1-AHs continue to be widely prescribed for AD despite conflicting evidence of their clinical efficacy [4,41,42,43]. In spite of systematic reviews which indicate that a histamine receptor blockade does not substantially improve AD-related pruritus or inflammation, and insufficient current evidence to recommend routine H1-AH therapy for AD, several studies have reported its potential clinical benefits. For example, cetirizine has been shown to reduce the need for topical corticosteroids, while combined H1R and H4R antagonism produced synergistic anti-inflammatory and antipruritic effects comparable to those of prednisolone [5,41,42]. Moreover, treatment with the AH fexofenadine resulted in a modest but significant reduction in AD-associated pruritus compared with a placebo, with improvement observed as early as the first day of treatment, suggesting that histamine acting through H1 receptors contributes, at least in part, to pruritus in AD. In our study, significantly higher AD severity indicators were observed in AD patients who discontinued AHs, while AD severity indicators significantly decreased (SCORAD, EASI and NRS) in those for whom AHs were introduced. These findings support the potential clinical utility of AH for select AD patients. Similarly, one study demonstrated that AHs may influence specific components of SCORAD, including significant patient-reported clinical improvements in pruritus and sleep disruptions, but without significant improvements in dermatologist-recorded SCORAD components (eczema extent and intensity). Still, this shows a favorable trend, which may reflect that histamine plays a (limited) role in these disease domains [5]. Several studies have supported the efficacy of AHs, particularly second-generation agents, in reducing pruritus and sleep disturbances in AD. Compared with first-generation AHs, second-generation agents have improved therapeutic indices and safety profiles, with minimal effects on cognitive function and sedation due to their limited penetration of the blood–brain barrier. Furthermore, their longer action duration allows less frequent administration. (Notably, cumulative use of first-generation AHs has been associated with an increased risk of cognitive impairment and dementia.) [5,44]. Most second-generation AHs exhibit higher selectivity for H1 receptors, resulting in fewer anticholinergic adverse effects (tachycardia, xerosis, delirium, etc.), which further supports their suitability for AD management [5]. Overall, in AD patients, AHs appear to be beneficial primarily through the reduction of pruritus and sleep disturbances, interrupting the itch–scratch cycle. In addition, their use may reduce the need for other topical therapies, such as corticosteroids, which are effective in controlling AD but may be associated with adverse effects (skin atrophy, exacerbation of pre-existing infections, contact dermatitis, etc.).

One of the main strengths of this study is its prospective design and that it is the first evaluation of melatonin levels in adult AD patients. Also, this is the first study which prospectively measured/followed salivary melatonin levels in adults with AD, and which simultaneously compared patients not using AH therapy with those in whom AH treatment was discontinued or introduced during follow-up. Salivary melatonin assessment offers several practical advantages over serum measurements: saliva collection is non-invasive, safer, and easier to perform repeatedly, and provides similar accuracy for the onset of melatonin action in low light [45]. These findings provide additional insight into the potential interplay between AH therapy, disease-related symptoms, and melatonin regulation in AD. Nevertheless, a few study limitations should be considered. Firstly, the relatively small sample size within individual study groups may have limited the statistical power and the generalizability of the findings. Larger, well-designed prospective studies would be needed to compare observations. Secondly, we only took one sample of saliva et each measurement timepoints. Multiple samples, taken in the evening, would have provide better insight into melatonin values. Overall, our findings support the potential use of H1-AHs as an adjunctive treatment in selected AD patients (e.g., for the management of pruritus and sleep disturbances when used in combination with standard topical anti-inflammatory therapy). Although current guidelines do not support H1-AH monotherapy for AD-associated pruritus, AHs may provide additional benefits through reduction of the itch–scratch cycle intensity and modulation of neural sensitization. Furthermore, the anti-inflammatory effects of topical corticosteroids may act synergistically with antihistaminic mechanisms, contributing to improved symptom control [46]. Further clinical studies are needed to better define the subgroup of AD patients who may benefit most from oral AH therapy and to clarify its long-term therapeutic potential.

4. Materials and Methods

4.1. Participant Recruitment and Eligibility

This prospective study was conducted at the Department of Dermatovenereology, University Hospital Center “Sestre Milosrdnice”, Zagreb, Croatia, between February 2025 and April 2026. and was approved by the Ethics Committee of the University Hospital Center “Sestre Milosrdnice”, Zagreb, Croatia, in January 2025 (number of protocol: 251-29-11/3-24-11). All patients included in the study gave their written informed consent to participate.

AD patients were enrolled in the study according to the following inclusion criteria: a diagnosis of AD based on the Hanifin and Rajka diagnostic criteria and age, ≥18 years [47,48,49]. We excluded AD patients who had received systemic corticosteroids and/or immunosuppressive, psychoactive, or biological therapy within the two weeks preceding study entry. We also excluded patients with a recent or current exacerbation of allergic asthma, rhinoconjunctivitis, or seasonal allergic rhinitis, as well as pregnant or breastfeeding women, smokers, and individuals working in shifts because of the potential impact on sleep quality. Additional exclusion criteria included the presence of oral diseases (oral candidiasis, other oral infections, oral lichen planus, oral injuries, or periodontal disease), severe chronic diseases (type 1 or type 2 diabetes mellitus, arterial hypertension, autoimmune diseases, or malignancies), the use of dentures or orthodontic appliances, and the inability to understand the nature and purpose of the study or the informed consent document. To exclude oral conditions and diseases, all participants underwent an oral examination performed by a dentist and/or a specialist for oral diseases.

In the end, the study included 57 adult patients with AD (41 women and 16 men), aged 18–53 years.

During the study, patients were allowed to use topical corticosteroid therapy (betamethasone) and regular skin care with emollients for the treatment of AD, provided that the treatment regimen remained stable throughout the entire study period (from baseline to the second study visit), without the introduction of any new medications that could affect AD. At the baseline visit, patients with AD received detailed information about the study, including comprehensive instructions on questionnaire completion, saliva collection, and sample storage. Saliva collection kits were provided during the visit together with written instructions covering restrictions regarding food, beverages, alcohol consumption, smoking, and appropriate oral hygiene before saliva collection.

4.2. Study Design

This prospective study, which included two assessments (T1 and T2) of the analyzed variables, comprised three groups of adult patients with AD: (I) patients not receiving AH therapy, (II) patients in whom AH therapy (bilastin or desloratadine) was discontinued, and (III) patients in whom AH therapy was introduced.

Patients in Group I remained without AH therapy throughout the study, including the two weeks preceding enrollment and the two-week interval between the first and second study visits, in accordance with current AD treatment guidelines, which do not recommend routine AH therapy. Patients in Group II had been receiving AH therapy before enrollment; however, they were instructed to discontinue treatment immediately after the first study assessment (T1) and to remain without AH therapy until the second study visit (T2), two weeks later. Patients in Group III initiated second-generation AH therapy immediately after the first study assessment and continued treatment for the subsequent two weeks until the second study visit.

At baseline (first study visit), all participants underwent the first assessment of salivary melatonin levels and clinical parameters related to AD severity and sleep. Most patients belonged to Group I and remained without AH therapy throughout the study (N=33). In Group II, second-generation AH therapy (bilastine or desloratadine) was discontinued after the first study visit (N=8). In Group III, second-generation AH therapy (bilastine or desloratadine, one tablet daily) was initiated immediately after the first study assessment and continued until the second study visit (N=16).

Two weeks after the baseline assessment, all participants underwent a second evaluation of the same study parameters. At both study visits, salivary melatonin samples were collected, and disease severity was assessed using the SCORing Atopic Dermatitis (SCORAD) index and the Eczema Area and Severity Index (EASI). Pruritus intensity was evaluated using the Peak Pruritus Numerical Rating Scale (Peak Pruritus NRS) [47,48,49]. Sleep quality was assessed only at the second study visit using the Pittsburgh Sleep Quality Index (PSQI), as this validated questionnaire evaluates sleep quality and sleep habits over the preceding month, corresponding to the overall study period from baseline to the second assessment [50,51].

We hypothesized that AD severity (as measured by SCORAD and EASI) is inversely proportional to salivary melatonin levels, and that there is an inverse correlation between disease severity indicators and itching intensity (NRS) and sleep indicators (PSQI) (less disease severity and less itching = better sleep). We also wanted to compare the results obtained before and after discontinuing AH therapy, i.e., whether this would cause a significant increase in AD severity (increased SCORAD, EASI) and a decrease in salivary melatonin, as well as whether a significant improvement in AD severity (decrease in SCORAD, EASI) and an increase in salivary melatonin would be seen after introducing AH therapy.

4.3. Salivary Melatonin Analysis

Saliva samples were collected between 22:00 and 00:00 h and stored in a refrigerator for a maximum of 3 days before being transferred to −80°C, where they remained until analysis. On the day of analysis, the samples were thawed and vortex-mixed at room temperature and then centrifuged at 1,500 × g for 10 min.

Melatonin concentrations were determined using a competitive enzyme-linked immunosorbent assay (ELISA) (Human Melatonin ELISA Kit, catalogue no. A327018, Antibodies.com Limited, Cambridge, United Kingdom) according to the manufacturer's instructions. The declared measuring range of melatonin was 15.63–1000 pg/mL and analytical sensitivity was 9.38 pg/mL. The intra-assay coefficient of variation (CV) was <7%, and the inter-assay CV was <9%.

4.4. Clinical Outcome Measures (Scores)

Disease severity was assessed using validated clinical scoring systems, including the SCORing Atopic Dermatitis (SCORAD) index and the Eczema Area and Severity Index (EASI) [47,48,49].

The SCORAD index assessed the extent of disease, intensity of clinical signs, and subjective symptoms, including pruritus and sleep disturbance during the previous three days. Subjective symptoms were evaluated using a visual analogue scale ranging from 0 to 10 (0 indicating no pruritus and 10 indicating maximum pruritus intensity). The total SCORAD score was calculated by combining the scores for disease extent, intensity of clinical signs, and subjective symptoms, with a maximum possible score of 103. Disease severity according to SCORAD was classified as mild AD (<25 points), moderate AD (25–50 points), and severe AD (>50 points) [48,49].

The EASI was used to evaluate disease extent across four anatomical regions (head and neck, trunk, upper extremities, and lower extremities) and to assess four clinical signs: erythema, induration/papulation, excoriation, and lichenification, each graded on a scale from 0 to 3. Disease severity according to the EASI score was categorized as: clear (0), mild (0.1–5.9), moderate (6.0–22.9), and severe (23.0–72) [47]. Unlike SCORAD, EASI does not include subjective symptoms such as pruritus and sleep disturbance.

At both study assessments, patients were asked to rate the intensity of pruritus experienced during the previous 24 hours using an 11-point numerical rating scale ranging from 0 (no itch) to 10 (worst itch imaginable). This measure represented the Peak Pruritus Numerical Rating Scale (Peak Pruritus NRS) [50].

Sleep quality was assessed only at the second study assessment using the PSQI, as this instrument evaluates sleep quality and sleep habits during the preceding month, corresponding to the period from baseline to the second study visit. The PSQI consists of seven components assessing subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep medication, and daytime dysfunction. A global PSQI score of >5 indicated clinically significant sleep disturbance [51].

4.5. Statistical Analysis

Salivary melatonin concentrations below the assay detection limit (15.63 pg/mL) were imputed as half the limit of detection (7.82 pg/mL) for statistical analyses. Such values were observed in 29/57 cases at the first sampling point and 26/57 cases at the second. The normality of data distribution was assessed using the Kolmogorov–Smirnov test. Correlations between salivary melatonin levels, disease severity, and sleep quality were evaluated using Spearman's rank correlation coefficient. Differences in melatonin levels and disease severity between the two timepoints were compared using the Wilcoxon signed-rank test. The effect size for the Wilcoxon test was calculated using the formula r=Z/√N and interpreted according to Cohen's criteria as small (0.10–0.29), medium (0.30–0.49), or large (>0.50). Differences among the three groups were analyzed using the Kruskal–Wallis test. Its effect size was quantified as Eta-squared using the formula η2=(H-k+1)/(n-k) and interpreted according to the squared values of Cohen's r criteria. The 95% confidence intervals (95% CI) for the median changes in melatonin levels and disease severity were estimated using bootstrapping with 1,000 resamples. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA).

5. Conclusions

Since our findings showed there is a significant negative linear correlation between AD severity and salivary melatonin levels, it is possible that melatonin supplementation for AD patients could improve their condition. AD patients who discontinued AHs after two weeks showed significantly increased AD severity and itch, and the group that started taking AHs showed significantly decreased AD severity. This supports the usefulness of AHs in the treatment of AD patients. Our findings thus support the potential use of H1-AHs as an adjunctive treatment for AD patients, particularly in combination with standard topical anti-inflammatory therapy. Although current guidelines do not mention/support H1-AH monotherapy for AD-associated pruritus/inflammation, AHs may provide additional benefits in the reduction of clinical AD features together with the anti-inflammatory effects of topical corticosteroids, as together they may act synergistically with antihistaminic mechanisms, contributing to improved AD symptom control. Further clinical studies are needed to better define the subgroup of AD patients who may benefit most from oral AH therapy and to clarify its long-term therapeutic potential.