This study investigates how different types of fasting affect cell health, specifically focusing on how mitochondria (the energy-producing parts of cells) are cleared and renewed. While standard intermittent fasting allows water intake, Ramadan dry fasting involves total restriction of both food and fluids from dawn to sunset.

Researchers want to see if the combined effects of fluid restriction and natural daily body rhythms during Ramadan trigger a stronger cellular cleanup process (called mitophagy) compared to standard 16:8 water-permitted fasting. The study will look at how these fasting habits change blood markers related to mitochondrial DNA and metabolic health across a 30-day period. This research will help determine if dry fasting offers distinct biological benefits for cellular renewal.

This is a prospective, single-centre, parallel-group, longitudinal observational cohort study conducted across Ramadan 2027. The study aims to evaluate the distinct molecular and metabolic trajectories of intermittent dry fasting compared to standard fluid-permitted time-restricted feeding (TRF).

Participants will be allocated to one of two parallel groups based on their self-selected, established fasting practices:

• Group A (Ramadan Dry Fasting): Observing complete fluid and nutrient deprivation from dawn (Fajr) to sunset (Maghrib) for 30 consecutive days.
• Group B (16:8 TRF): Practicing a 16-hour daily fasting window with water/fluids permitted, observed concurrently during the same calendar period.

Participants in both groups will be pairwise-matched based on age and BMI at screening. Blood samples will be collected from all participants at five distinct milestones:

• T0: Pre-Ramadan baseline (Day -3)
• T1: Acute induction phase (Day 7)
• T2: Mitochondrial turnover phase (Day 14)
• T3: Saturation plateau / primary endpoint (Day 30)
• T4: Post-Ramadan recovery phase (Day 44)

Peripheral blood mononuclear cells (PBMCs) and serum will be isolated from the collected blood. Laboratory analyses will measure mitochondrial DNA (mtDNA) copy number changes via qPCR, alongside a quantitative gene expression panel evaluating key regulators of mitophagy (such as PINK1 and Parkin), mitochondrial biogenesis, and general autophagy. Secondary evaluations include routine serum metabolic indicators, inflammatory markers, and dietary monitoring to evaluate the physiological pathways predicted by computational systems pharmacology.

Inclusion Criteria:

• Common Criteria (Both Arms):
• Age 18-50 years
• BMI 18.5-30.0 kg/m²[cite: 1]
• Non-smoker for at least 12 months[cite: 1]
• No chronic disease requiring regular prescription medication[cite: 1]
• Stable sleep-wake schedule (self-reported sleep midpoint deviation less than 90 minutes)[cite: 1]
• Willing and able to attend all five study visits at the specified times[cite: 1]

Arm A Specific (Ramadan Dry Fasting):

- Documented religious commitment and intention to complete all 30 days of Ramadan 2027 fasting[cite: 1]

Arm B Specific (16:8 TRF Control):

• Confirmed practice of 16:8 TRF (fasting window 16 hours or more per day) throughout the study period[cite: 1]
• Not observing Ramadan dry fasting during the study period[cite: 1]

Exclusion Criteria:

• Established type 1 or type 2 diabetes (HbA1c 6.5% or higher or pharmacological antidiabetic therapy)[cite: 1]
• Severe gastrointestinal, cardiovascular, hepatic, or renal disease (eGFR less than 60 mL/min/1.73m²)[cite: 1]
• Active cancer or chemotherapy within five years[cite: 1]
• Use of metformin, statins, or exogenous antioxidant supplements (vitamins C, E, NAC, CoQ10) within 30 days of screening[cite: 1]
• Pregnancy, lactation, or planned conception during the study period[cite: 1]
• Participation in another clinical study within three months[cite: 1]
• Inability to provide written informed consent in Arabic or English[cite: 1]

The study population consists of healthy adult volunteers aged 18 to 50 with a BMI between 18.5 and 30.0 kg/m². The cohort represents individuals who are either observing the religious dry fast of Ramadan or concurrently practicing standard 16:8 time-restricted feeding. Participants are recruited from the academic and local communities of Bahçeşehir Cyprus University and Near East University in Nicosia, Northern Cyprus[cite: 1]. The sample includes both males and females who do not smoke, have stable sleep-wake cycles, and are free from chronic diseases requiring regular prescription medications[cite: 1].