Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is the most common cause of chronic liver disease in children and adolescents. Lifestyle factors are modifiable risk factors that may play a key role in both the prevention and management of the disease. However, existing data on the association between lifestyle and MASLD in pediatric populations are limited and often focus on isolated aspects such as diet or physical activity, with little attention given to other parameters like sleep habits. The aim of the present study is to comprehensively investigate the association between lifestyle factors, including dietary habits, physical activity, sedentary activities, and sleep habits, and the presence of MASLD in a sample of 224 children and adolescents with overweight or obesity. The study will include newly diagnosed MASLD patients compared to matched controls without the disease. A wide range of assessments will be conducted, including anthropometric measurements, body composition analysis, liver elastography, biochemical testing, and standardized lifestyle questionnaires. This study seeks to fill important research gaps and explore potential associations between lifestyle habits and pathophysiological markers involved in the onset and progression of MASLD.

The study sample will consist of 224 children and adolescents aged 10-18 years with overweight or obesity, including individuals newly diagnosed with Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and matched controls without the disease.

The diagnosis of MASLD will be based on the criteria established by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN): elevated alanine aminotransferase (ALT) levels >45 IU/L and/or evidence of hepatic steatosis on ultrasound, in the absence of other causes of liver steatosis or liver injury. Where available, liver biopsy results will be included in the participants' medical records.

Participants in the control group will be matched to the MASLD group based on age, sex, body mass index (BMI), and pubertal stage. Eligible controls must present with normal liver biochemistry, no ultrasound evidence of hepatic steatosis, normal glucose metabolism, and stable body weight and lifestyle behaviors during the past year.More specifically, the study will include:

1. Medical and family history: Detailed documentation of personal and family medical history, current medications, and birth-related data (e.g., gestational age, birth weight, breastfeeding history).
2. Demographic and socioeconomic data: Collected from both parents and participants, including education level, employment status, ethnicity, and household composition.
3. Anthropometric measurements and body composition: Body weight, height, BMI, waist and hip circumference will be recorded. Body composition will be assessed using bioelectrical impedance analysis (Tanita MC 780 MA), with standardized conditions (e.g., fasting state, no intense physical activity, no caffeine intake).
4. Blood pressure: Measured in a seated position with an electronic sphygmomanometer on the right arm. Three measurements will be taken, and the mean value will be recorded.
5. Liver imaging: All participants will undergo abdominal ultrasound for assessment of hepatic steatosis, and those with MASLD will undergo liver elastography (Shear Wave Elastography) within 3 months of enrollment to evaluate liver fibrosis.
6. Laboratory testing: After 8-12 hours of fasting, blood samples will be collected to determine standard biochemical profile, including liver enzymes (ALT, AST, GGT), bilirubin, lipids (total cholesterol, HDL, LDL, triglycerides), glucose, insulin, kidney function, electrolytes, and total protein, hematologic markers (complete blood count and erythrocyte sedimentation rate), inflammation and oxidative stress biomarkers, and oxidative stress markers. Serum, plasma, and urine samples will be stored at -80°C for future analyses. DNA will also be extracted for potential genotyping.
7. Fatty Liver Index (FLI): FLI will be calculated using waist circumference, BMI, triglyceride levels, and GGT to assess the risk of hepatic steatosis.
8. Dietary assessment: Each participant will complete four 24-hour dietary recalls (three weekdays and one weekend day). Nutrition data will be analyzed for energy, macro- and micronutrient intake using Nutritionist Pro (v2.2), with additional assessment of food groups and dietary patterns. Adherence to the Mediterranean Diet will be assessed using the KIDMED index.
9. Physical activity assessment: Performed using the Self-Administered Physical Activity Checklist (SAPAC), validated for the Greek pediatric population. It will be completed four times, in conjunction with the dietary recalls.
10. Sleep habits assessment: Evaluated with the Pediatric Daytime Sleepiness Scale (PDSS), along with additional questions about sleep duration, naps, and rest habits on weekdays and weekends.

Inclusion Criteria:

• Age: Children and adolescents aged 10 to 18 years, of Caucasian ethnicity, regardless of sex or nationality.

• Overweight or Obesity: Children/adolescents with overweight or obesity according to World Health Organization (WHO) criteria for ages 5-19.

• Newly Diagnosed Metabolic Dysfunction-Associated Steatotic Liver Disease: Diagnosis will be established in participants who meet all of the following criteria:

  • Elevated ALT levels (>45 IU/L), according to the European Society for Paediatric Gastroenterology, Hepatology and Nutrition, and/or evidence of hepatic steatosis on liver ultrasound.
  • Exclusion of other causes of liver damage or steatosis, including negative for HBsAg, anti-HIV, and anti-HCV antibodies, no autoimmune hepatitis, no viral infections, systemic or metabolic diseases, no celiac disease, chronic kidney disease, normal ceruloplasmin levels (to rule out Wilson's disease), no hepatotoxic medications.

Exclusion Criteria:

• Type 1 diabetes mellitus
• Alcohol consumption
• Malnutrition or parenteral nutrition
• Thyroid function
• Recent changes in diet and physical activity habits aimed at weight loss and/or improvement of health parameters in newly diagnosed children and adolescents

224 children and adolescents with overweight/ obesity and/or Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD)