1. Body Weight and Height Participants will fast overnight prior to measurement. Body weight will be measured in the morning using a calibrated Seca digital scale, with participants wearing light clothing and no shoes. Height will be measured using a Seca 217 stadiometer, with participants standing barefoot.
2. Resting Energy Expenditure (REE) REE will be measured via indirect calorimetry using a respiratory hood system (Cosmed). Participants will report to the laboratory after an overnight fast, lie supine on a flat bed, and have the hood placed over their head. Oxygen consumption and carbon dioxide production will be recorded for 40 minutes, with the final 10 minutes used for analysis. The system will be quality-checked regularly using a turbine test, and monthly validation will be conducted via an alcohol combustion test.
3. Total Energy Expenditure (TEE)
TEE will be assessed using two complementary methods:
Doubly Labeled Water (DLW) Method: Urine samples from the DLW subset will be stored at -20 °C and shipped on dry ice to Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. Isotope ratios will be analyzed using a Liquid Water Isotope Analyzer (ABB). CO₂ production will be calculated using the latest equation from Speakman et al. (2021, Cell Reports Medicine), and TEE will be estimated using the Weir equation.
Metabolic Chamber: TEE will also be measured using a whole-body metabolic chamber (Maastricht Instruments, Netherlands), which continuously tracks oxygen consumption and carbon dioxide production. Participants will remain in the chamber for 48 hours, engaging in routine daily activities. Data from the second day will be analyzed to estimate TEE.
4. Physical Activity Physical activity will be assessed using a GT3X accelerometer worn on the hip for 15 consecutive days. The device should not be worn during bathing or swimming. Data from the first day and any day with \<12 hours of wear time will be excluded. A valid dataset requires at least two weekdays and two weekend days of usable data.
5. Body Composition
Body composition will be measured using the following techniques:
Magnetic Resonance Imaging (MRI; Philips Multiva 1.5T) Dual-energy X-ray Absorptiometry (DXA; HOLOGIC Horizon Wi, USA) Bioelectrical Impedance Analysis (BIA; TANITA MC-980, Japan) Parameters to be assessed include total and regional fat mass, lean mass, and fat percentage across the limbs and trunk. MRI will be used for whole-body fat distribution analysis by trained technicians.
6. Mental Health Assessment The Symptom Checklist-90 (SCL-90) will be used to evaluate psychological well-being. This tool measures ten dimensions: somatization, obsessive-compulsive traits, interpersonal sensitivity, depression, anxiety, hostility, phobia, paranoid ideation, psychosis, and additional symptoms. Scores for each dimension and overall symptom severity will be calculated.
7. Food Intake and Hunger Assessment Meals will be designed based on the energy distribution guidelines from the Chinese Dietary Guidelines. After a standardized lunch, participants will receive a test meal (packaging removed) containing predefined macronutrient and energy levels five hours later. Food intake will be ad libitum, and hunger levels will be assessed based on behavioral indicators. Blood samples (4 mL) will be collected at baseline (pre-dinner), and 15, 30, and 60 minutes post-dinner for appetite-related hormones analysis such as ghrelin and PYY.
8. Sleep Quality Monitoring Sleep will be monitored using the Heartcare Youhu Pro AI Precision Health Management System, which tracks total sleep duration, sleep efficiency, cycle durations, heart rate, and respiratory patterns. These metrics will be used to assess sleep quality.
9. Cognitive Function Assessment
Cognitive performance will be evaluated using:
Mini-Mental State Examination (MMSE): Assesses orientation, memory, attention, language, and visuospatial function. Scores of 25-30 indicate normal function; 20-24 mild, 10-19 moderate, and \<10 severe cognitive impairment.
Montreal Cognitive Assessment (MoCA): Screens for impairment across eight cognitive domains. A score ≥26 is considered normal.
10. Non-Invasive Brain Function Monitoring Functional Near-Infrared Spectroscopy (fNIRS) will be used to assess brain activity by measuring changes in oxyhemoglobin and deoxyhemoglobin concentrations. We may use the BS2000 near-infrared imaging system for this purpose.
Assessment Areas: Prefrontal cortex, motor cortex, frontal pole, dorsolateral prefrontal cortex, primary motor/somatosensory cortex, premotor and supplementary motor cortex-on both healthy and affected sides.
Key Indicators:HbO, HbR, and HbT concentration changes during a 0-40 sec task window. 3D topological maps (HbO beta values). Activation levels at rest. Functional connectivity (HbO).
11. Gait Analysis Laboratory
A 3D markerless motion capture system (Xiangneng-Dongxi) will be used for gait analysis. This non-invasive system captures:
Spatiotemporal Parameters: Step time, frequency, speed, stride length/width, and symmetry Kinematic Parameters: Joint angles (pelvis, hip, knee), motion curves, ground reaction forces, and joint torques
12. Gut Microbiota and Blood Metabolomics Mid-portion fecal samples and 5 mL blood samples will be collected. Fecal samples will undergo non-targeted microbiome analysis at Novogene, while blood samples will be analyzed at Biotree Bioscience for metabolomic profiling, including appetite-related hormones such as ghrelin and PYY.
