The purpose of this observational study is to systematically evaluate how a 12-month multimodal psychiatric treatment program affects sleep quality, inflammatory and hormonal biomarkers, eating disorder psychopathology, and health-related quality of life in adolescent girls (aged 10-18 years) diagnosed with anorexia nervosa.

The main questions it aims to answer are:

• Do adolescent girls with anorexia nervosa have measurable changes in sleep patterns (total sleep time, sleep efficiency, REM sleep, deep sleep) compared to age- and sex-matched published values, and do these improve after 12 months of psychiatric treatment?
• Are specific body markers (like NLR, CRP, vitamin D, and lipid profile) linked to how severe sleep problems, eating disorder symptoms, and treatment results are in this group?
• To assess these outcomes, researchers will compare participants' baseline measurements to their own 12-month follow-up measurements to determine whether multimodal psychiatric treatment produces clinically meaningful improvements in sleep architecture, biomarker profiles, psychopathology, psychological flexibility, and quality of life.

Participants will:

• Take-home sleep recordings with a wireless device (NOX A1) in their usual environment at the start and after 12 months of treatment.
• Complete validated self-report questionnaires assessing sleep quality (PSQI), daytime sleepiness (ESS-CHAD for children and adolescents), eating disorder symptoms (EDE-Q), depression, anxiety, and stress (DASS-21), psychological flexibility (AFQ-Y8), and health-related quality of life (KIDSCREEN-52) at baseline and 12-month follow-up.
• Give blood samples at the start and after 12 months for testing of inflammation markers, hormone levels, lipids, and anthropometric measurements.
• Take part in a Day Hospital Program at the Department of Psychiatry, University Hospital of Split (KBC Split), Croatia, which includes Acceptance and Commitment Therapy, Cognitive Behavioral Therapy, individual counselling, family work, and medication if needed.

Anorexia nervosa (AN) is a severe psychiatric disorder defined by restrictive food intake, significant weight loss, distorted body image, and an intense fear of weight gain, resulting in a three times higher mortality rate among individuals with AN than in the general population. The peak incidence occurs at 14 years of age, with a prevalence of approximately 1.7% among adolescent girls. While early intervention during adolescence is associated with improved outcomes, chronic illness persisting into adulthood often results in recovery rates of only 20-30%.

Sleep disturbances are a clinically significant yet systematically underinvestigated aspect of AN. Almost 50% of individuals with AN report sleep difficulties, irrespective of disorder subtype. A 2024 meta-analysis demonstrated that patients with AN exhibit changes in sleep architecture resulting in significantly shorter total sleep time (TST), reduced sleep efficiency (SE), prolonged wake after sleep onset (WASO), increased N1 non-REM sleep, and reduced REM sleep compared to healthy controls, and especially reduced slow-wave sleep (SWS). Reduced quality of sleep is associated with poorer treatment compliance, increased therapy discontinuation, and a reduced probability of recovery. Notably, weight restoration alone does not regularly normalize sleep architecture, indicating that sleep disturbances require targeted clinical attention independent of nutritional rehabilitation.

The mechanisms underlying sleep disturbances in AN remain unclear. Malnutrition induces hormonal alterations, including elevated ghrelin and orexin-A, decreased leptin, and increased cortisol, that, combined, promote wakefulness and disrupt circadian rhythms. AN is uniquely associated with a morning chronotype, in contrast to most other psychiatric conditions, which are linked to eveningness; this distinction has recently been confirmed by Mendelian randomization studies. Comorbid depression and anxiety, which are highly prevalent in AN, further induce sleep difficulties via overlapping neurobiological pathways.

The majority of current literature has focused on adult populations. Adolescent-specific data remain limited and methodologically inconsistent, with most studies relying on questionnaire-based measures or laboratory polysomnography, both of which are subject to first-night effects.

To date, no published study has combined objective home-based sleep measurement with comprehensive longitudinal biomarker assessment specifically in early-to-mid-adolescent girls with AN. The increasing frequency of pre-menarchal presentations in clinical practice further indicates the necessity for pediatric-focused evidence. In addition to sleep, peripheral biomarkers in AN remain incompletely characterized in adolescents. Data from adult cohorts indicate elevated inflammatory cytokine levels, low-grade systemic inflammation, altered thyroid and prolactin function, dyslipidemia, and reduced 25-hydroxyvitamin D. The neutrophil-to-lymphocyte ratio (NLR), a readily available marker of physiological stress and systemic inflammation, is dysregulated in adult AN. However, the applicability to the adolescent population and their evolution over time with treatment remain insufficiently investigated.

This study identifies two primary gaps: the absence of objective home-based sleep data and the lack of longitudinal biomarker trajectories specifically in adolescent girls with AN undergoing structured psychiatric treatment.

The study will be conducted at the Day Hospital for Children and Adolescents, Department of Psychiatry, University Hospital of Split (KBC Split), Croatia. Participants will be consecutive female patients aged 10-18 years diagnosed with anorexia nervosa (ICD-10: F50.0, F50.1, F50.9) referred to and treated by a child and adolescent psychiatrist at KBC Split.

At enrollment, a detailed history will be collected, including developmental, family, and menstrual history (menarche status and menstrual irregularities), and social background. Parental heteroanamnestic data will be collected separately. Physical examination will include vital signs (heart rate, blood pressure, body temperature) and anthropometric measures (body weight, height, BMI).

All assessments are conducted at two time points:

T0 - Baseline (study enrollment, prior to or at initiation of Day Hospital treatment) T1 - 12-month follow-up (after one year of structured multimodal treatment)

At both time points, participants will undergo:

OBJECTIVE SLEEP ASSESSMENT Home polysomnography using the NOX A1 portable wireless device (Nox Medical). The NOX A1 is a full-channel ambulatory polysomnographic system that eliminates the discomfort associated with traditional in-laboratory PSG and enables sleep recording under naturalistic home conditions. The device records electroencephalography (EEG), electrooculography (EOG), chin electromyography (EMG), electrocardiography (ECG), respiratory effort (thoracic and abdominal bands), nasal airflow, pulse oximetry, body position, and actigraphy. Sleep staging will follow AASM 2023 scoring criteria. Participants will self-apply the device at home following standardized written and verbal instructions. Derived variables: TST (minutes), SE (%), SOL (minutes), WASO (minutes), N1%, N2%, N3% (SWS), REM%, REM latency (minutes), arousal index.

SUBJECTIVE SLEEP AND SLEEPINESS ASSESSMENT Pittsburgh Sleep Quality Index (PSQI) - 19-item self-report assessing sleep quality over the previous month; global score >5 indicates clinically poor sleep quality.

Epworth Sleepiness Scale for children and adolescents (ESS-CHAD) - 8-item self-report measure of daytime sleepiness; score >10 indicates excessive daytime sleepiness; participant version completed by the adolescent; ESS-CHAD for parent/caregiver version completed independently by parent or legal guardian (Croatian validated version) Sleep habits questionnaire - structured assessment of sleep timing, duration, screen use, and sleep behavior

EATING DISORDER PSYCHOPATHOLOGY Eating Disorder Examination Questionnaire (EDE-Q) - 28-item self-report; global score and four subscales (Restraint, Eating Concern, Shape Concern, Weight Concern); higher scores indicate greater eating disorder psychopathology EMOTIONAL AND PSYCHOLOGICAL ASSESSMENT Depression Anxiety Stress Scale - 21 items (DASS-21) - self-report measure of depression, anxiety, and stress symptom frequency and severity over the preceding week; Subscale scores doubled per standard scoring to yield scores comparable to the full DASS-42 Avoidance and Fusion Questionnaire for Youth - 8 items (AFQ-Y8) - self-report measure of psychological inflexibility, experiential avoidance, and cognitive fusion in children and adolescents

QUALITY OF LIFE KIDSCREEN-52 - 52-item health-related quality of life instrument for children and adolescents (10 dimensions: physical well-being, psychological well-being, moods and emotions, self-perception, autonomy, parent relations, social support and peers, school environment, social acceptance, financial resources); both participant self-report version and parent proxy version were completed independently

LABORATORY BIOMARKER ASSESSMENT Blood samples collected under standardized fasting conditions (minimum 8-hour fast) at both time points: Inflammatory markers: complete blood count with differential, neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP, mg/L).

Hormonal and nutritional markers: free thyroxine (fT4), free triiodothyronine (fT3), prolactin, 25-hydroxyvitamin D (25OH-D).

Metabolic markers: total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides TREATMENT DOCUMENTATION

For each participant, the following will be systematically recorded throughout the 12-month treatment period:

• Pharmacotherapy: agent, dose, duration, and any modifications
• Psychotherapy: modality, number of sessions attended, adherence
• Physical and psychiatric status at each clinical contact
• Adverse events and side effects
• Treatment discontinuation and reasons TREATMENT PROGRAMME

All participants are enrolled in the structured Day Hospital Program for Children and Adolescents at the Department of Psychiatry, KBC Split. The program comprises:

1. Acceptance and Commitment Therapy (ACT) combined with Cognitive Behavioral Therapy (CBT) - group and individual format targeting cognitive distortions, body image, emotions, and values-based behavior change
2. Supportive individual psychotherapy - addressing motivation for recovery, self-esteem, and interpersonal functioning
3. Family-based work and parent sessions - psychoeducation, family communication, and caregiver support
4. Psychoeducational interventions - nutrition, body development, sleep hygiene, and emotion regulation
5. Pharmacotherapy - prescribed where clinically indicated by the treating child and adolescent psychiatrist; type, dose, and duration recorded as covariates

STATISTICAL ANALYSIS PLAN

All statistical analyses will be performed using IBM SPSS Statistics (version 29.0 or later) and R (version 4.3.0 or later). A two-tailed significance level of p < 0.05 will be applied throughout. Given the exploratory and hypothesis-generating nature of this study, both uncorrected and Bonferroni-corrected p-values will be reported for multiple comparisons. Continuous variables: mean ± standard deviation (SD) for normally distributed data; median and interquartile range (IQR) for non-normally distributed data. Normality will be assessed using the Shapiro-Wilk test. Categorical variables: frequencies and percentages.

WITHIN-SUBJECT CHANGE (T0 TO T1) Paired-samples t-test (parametric) or Wilcoxon signed-rank test (non-parametric) will be used to assess the change in continuous outcome measures from baseline to 12 months follow-up. Effect sizes will be reported as Cohen's d (parametric) or rank-biserial correlation r (non-parametric). Spearman's rank correlation will be used to examine associations among sleep parameters, biomarker levels, psychopathological scores, and quality-of-life measures at both time points and for change scores (ΔT1-T0). Multivariable linear regression will be applied to identify independent predictors of: (1) sleep architecture parameters at baseline; (2) change in EDE-Q global score at 12 months. Covariates will include age, BMI z-score, illness duration, depression score (DASS-21), and pharmacotherapy status. The assumptions of linearity, independence, homoscedasticity, and the absence of multicollinearity will be verified prior to modeling. Longitudinal changes in biomarker panels will be examined using repeated-measures analysis or mixed-effects models, as applicable, with pharmacotherapy as a between-subject factor.

SAMPLE SIZE CONSIDERATIONS This is a single-center prospective cohort study with consecutive enrollment. A formal a priori power calculation is limited by the absence of comparable pediatric polysomnographic data in AN. Based on estimated annual referral rates at the KBC Split Day Hospital Program. A sample of 30-50 eligible participants is anticipated over the enrollment period. The study is, therefore, appropriately characterized as exploratory and hypothesis-generating, with findings intended to inform the design of adequately powered future multi-center studies. The study will be conducted in accordance with the Declaration of Helsinki and applicable Croatian and European Union regulations on clinical research and data protection (GDPR).

Ethical approval will be obtained from the Ethics Committee of the University Hospital of Split (KBC Split) and the Ethics Committee of the University of Split School of Medicine prior to enrollment of any participant. Written informed consent will be obtained from each participant's parent or legal guardian. Written assent will be obtained from each participant. Participation is voluntary, and withdrawal at any time will not affect the quality of clinical care provided. All data will be pseudonymized and stored in accordance with institutional data protection protocols.

Inclusion Criteria:

1. Female sex assigned at birth

2. Age between 10 and 18 years (inclusive) at the time of study enrollment

3. First-time diagnosis of anorexia nervosa according to ICD-10 criteria at the time of enrollment, including:

   • Anorexia nervosa (F50.0)
   • Atypical anorexia nervosa (F50.1)
   • Eating disorder, unspecified (F50.9) with predominant restrictive or anorexic presentation

4. Currently under the care of a child and adolescent psychiatrist at the Department of Psychiatry, University Hospital of Split (KBC Split), Croatia

5. Enrolled in or eligible for the Day Hospital Program for Children and Adolescents at the Department of Psychiatry, KBC Split

6. Ability to read and understand Croatian language sufficiently to complete self-report questionnaires

7. Parent or legal guardian willing and able to provide written informed consent prior to any study procedure

8. Participant willing and able to provide written assent prior to any study procedure

9. Participant and parent/guardian willing to undergo all study procedures at both time points, including:

   • Home polysomnography (NOX A1) at baseline and 12-month follow-up
   • Completion of all self-report questionnaires at baseline and 12-month follow-up
   • Fasting blood sampling at baseline and 12-month follow-up

Exclusion Criteria:

1. Age younger than 10 years or older than 18 years at the time of enrollment

2. Male sex assigned at birth

3. Previous diagnosis of anorexia nervosa prior to current enrollment (i.e., recurrent or relapsing AN - this study enrolls first-diagnosis cases only)

4. Anorexia nervosa currently in clinical remission at the time of enrollment

5. Comorbid psychiatric diagnosis of any of the following:

   • Intellectual disability (any severity, ICD-10: F70-F79)
   • Autism spectrum disorder (ICD-10: F84)
   • Schizophrenia spectrum or other psychotic disorder (ICD-10: F20-F29)
   • Bipolar affective disorder, any type (ICD-10: F31)
   • Substance use disorder, any substance (ICD-10: F10-F19)

6. Severe or chronic somatic illness documented in medical history, including but not limited to:

   • Central nervous system disorders (epilepsy, acquired brain injury, neurodegenerative disease, ICD-10: G00-G99)
   • Chronic inflammatory or autoimmune disease (e.g., inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis)
   • Primary endocrine disorder independent of AN (e.g., primary hypothyroidism, type 1 or type 2 diabetes mellitus, congenital adrenal hyperplasia)
   • Active or history of malignancy
   • Chronic renal or hepatic disease

7. Use of any medication known to significantly alter sleep architecture or inflammatory biomarker levels that was initiated prior to study enrollment and is unrelated to the study treatment program, including:

   • Benzodiazepines or z-drugs (zopiclone, zolpidem)
   • Antipsychotic medications
   • Systemic corticosteroids
   • Immunosuppressive agents
   • Melatonin or melatonin receptor agonists Medications initiated as part of the Day Hospital treatment program after enrollment will be documented and included as covariates in statistical analyses and do not constitute an exclusion criterion.

8. Presence of an active somatic condition at the time of enrollment requiring acute inpatient medical treatment (e.g., severe electrolyte imbalance requiring intravenous correction, acute cardiac arrhythmia) that would preclude safe participation in the Day Hospital Program

9. Physical or cognitive inability to cooperate with home polysomnography device placement or self-report questionnaire completion, as judged by the treating child and adolescent psychiatrist

10. Simultaneous participation in another interventional clinical trial that could influence sleep parameters, nutritional status, inflammatory markers, or psychiatric outcomes.

11. Absence of written informed consent from parent or legal guardian, or absence of written assent from the participant

Female patients aged 10-18 years with a first-time diagnosis of anorexia nervosa (ICD-10: F50.0, F50.1, F50.9) enrolled consecutively at the Day Hospital for Children and Adolescents, Department of Psychiatry, University Hospital of Split (KBC Split), Croatia. Participants must be medically stable for Day Hospital treatment and able to complete home polysomnography and self-report questionnaires in Croatian. Excluded are patients with intellectual disability, autism spectrum disorder, psychotic or bipolar disorder, substance use disorder, chronic somatic illness affecting inflammatory markers, or prior AN diagnosis.

Individual participant data will not be shared because the study involves sensitive adolescent related clinical, psychological and metabolic data, and current approvals cover analysis only within the research team. De-identified aggregate data my be shared in publications or upon reasonable request.