BIOANCIENT_EX: Effects of a Physical Exercise Program in Centenarians of the BIOANCIENT Cohort: A Randomized Clinical Trial.

Physical Function: Assessed using the Short Physical Performance Battery (SPPB), which combines balance, gait velocity, and leg strength as a single score on a 0 (worst) to 12 (best scale).

The EQ VAS ranges from 0 (worst imaginable health) to 100 (best imaginable health). Higher scores indicate better health status. The EQ-5D-5L descriptive system is a standardized instrument for measuring health-related quality of life. It comprises 5 dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension has 5 levels of severity: no problems, slight problems, moderate problems, severe problems, and extreme problems. This descriptive system is converted into a single utility index score using a country-specific value set. Index scores typically range from less than 0 (where 0 represents death and negative values represent states worse than death) to 1 (representing perfect health). Higher scores indicate better health states.

The EQ Visual Analogue Scale (EQ VAS) is a component of the EQ-5D-5L questionnaire that records the respondent's self-rated health on a vertical visual analogue scale. Scores range from 0 to 100, where 0 represents 'the worst health you can imagine' and 100 represents 'the best health you can imagine'. Higher scores indicate a better self-perceived health status.

Barthel Index of independence during activities of daily living (ADLs) ranges from 0 (severe functional dependence) to 100 (functional independence).

Cognitive Function assessed with Mini-Mental State Examination (MMSE) is a 30-point tool designed to assess global cognitive function and screen for cognitive impairment, particularly in older adults. It evaluates several core areas of cognitive functioning, with higher scores indicating better cognitive performance, i.e. 30 (best) and 0 (worst).

The 11 tasks in the MMSE are generally categorized into the following areas of cognitive functioning: Orientation (10 points), Registration (3 points), Attention and Calculation (5 points); Recall (3 points): .Language and Praxis (9 points).

Muscle Strength assessed by handgrip strength using a dynamometer (Takey Physical Fitness Test) measured in kilograms. The minimum recorded measurement is 5 kg and the maximum is 100 kg. Grip strength is a powerful biomarker of aging and overall muscle strength. Low handgrip strength is associated with increased risks of cardiovascular disease, stroke, type 2 diabetes, and premature mortality.

Lower Limb Muscle Strength. Isometric lower limb muscle strength assessed using a Hoggan dynamometer. Knee extensor strength and hip flexor strength will be measured with participants in a standardized seated position, starting with the knee flexed (90º). Force will be recorded in kilograms (kg). Higher values indicate greater muscle strength.

Cognitive function assessed using the Clock Drawing Test (CDT), in which participants are asked to draw a clock showing a specified time. The test evaluates executive function, visuospatial ability, and planning. Performance will be scored using a standardized scoring system, with higher scores indicating better cognitive performance. Results range from 0 (worst) to 10 (best).

Nutritional Status assessed using the Mini Nutritional Assessment - Short Form (MNA-SF), a rapid, validated 6-item screening tool designed to identify older adults (aged 65+) who are malnourished or at risk of malnutrition. It is widely used in hospitals, nursing homes, and community settings to initiate early nutritional interventions. Scoring from 14 points (better) and 0 points (worse):12-14 points: Normal nutritional status; 8-11 points: At risk of malnutrition and; 0-7 points: Malnourished.

Risk of sarcopenia assessed using the SARC-F questionnaire, a five-item self-reported screening tool evaluating strength, assistance in walking, rising from a chair, climbing stairs, and falls. Total scores range from 0 to 10, with higher scores indicating greater risk of sarcopenia and functional decline.

Depressive symptoms assessed using the Geriatric Depression Scale (GDS-15), a 15-item self-reported questionnaire designed to screen for depression in older adults. Scores range from 0 to 15, with higher scores indicating more severe depressive symptoms. Scores of 0-4 are considered normal, 5-9 suggest mild depression, and 10-15 indicate moderate to severe depression.

Mobility and functional performance assessed using the Timed Up and Go (TUG) test. Participants are instructed to stand up from a standard chair, walk a distance of 3 meters, turn around, walk back to the chair, and sit down. The total time to complete the task is recorded in seconds. Lower times indicate better mobility and functional performance.

Times greater than or equal to 12 seconds are commonly associated with increased risk of falls in older adults.

Bioimpedance Analysis (BIVA - Bioelectrical Impedance Vector Analysis): Whole-body bioelectrical impedance vector analysis (BIVA) assessed using a phase-sensitive bioimpedance device (e.g., BIVA Pro). Raw bioelectrical parameters including resistance (R, ohms) and reactance (Xc, ohms) will be measured at 50 kHz and normalized by height (R/H, Xc/H).

BIVA provides a qualitative and semi-quantitative assessment of body composition, including hydration status and body cell mass, without relying on predictive equations. Vector displacement patterns will be interpreted as follows:

Vector shortening (↓R/H, ↓Xc/H): fluid overload or increased hydration Vector lengthening (↑R/H, ↑Xc/H): dehydration or reduced fluid content Increased phase angle: better cellular integrity and nutritional status Decreased phase angle:

impaired cellular health or frailty.

Proteomic biomarkers assessed through the identification and quantification of circulating proteins associated with aging and frailty using high-throughput proteomic techniques. Protein expression levels will be measured in biological samples (e.g., plasma or serum), and relative or absolute concentrations will be analyzed.

Total number of unintentional falls experienced by the participant during the 12 months prior to the assessment. A fall is defined as an unexpected event in which the participant comes to rest on the ground, floor, or lower level. Data will be collected via self-report calendar.

Assessment of the participant's fear of falling, categorized as a dichotomous outcome (Yes or No). Participants are asked the single standard question: "Are you afraid of falling?". A response of "Yes" indicates the presence of fear, while "No" indicates its absence.

Pain intensity will be assessed using a standard Visual Analog Scale (VAS). The scale consists of a 10 cm horizontal line, where the left endpoint (0) represents "no pain" and the right endpoint (10) represents "worst imaginable pain". Participants mark the point on the line that best represents their current pain level. Higher scores indicate greater pain severity.

Polypharmacy assessed as the concurrent use of multiple medications. The total number of regularly prescribed medications will be recorded for each participant. Polypharmacy will be defined as the use of five or more medications (≥5). The outcome will be analyzed both as a continuous variable (number of medications) and as a categorical variable (presence vs absence of polypharmacy). Higher values indicate greater medication burden.

Prior and Concomitant Medication History; Baseline assessment of chronic pharmacological exposure. Participants report lifetime use (lasting >1 year) of 27 key drug classes (Yes/No), including cardiovascular, endocrine, respiratory, analgesic, anti-infective, neuropsychiatric, gastrointestinal, bone, and immune therapies. Long-term historical medications are also captured via open text. Reported as counts and percentages.

Comorbidity burden assessed using the Cumulative Illness Rating Scale for Geriatrics (CIRS-G), which evaluates the severity of chronic medical conditions across 14 organ systems. Each system is rated on a 5-point scale (0 = no problem to 4 = extremely severe impairment). The total score ranges from 0 to 56, with higher scores indicating greater comorbidity burden.

Evaluation of participant medical history and current health status classified according to the International Classification of Diseases, 10th Revision (ICD-10). Diagnoses will be extracted from electronic medical records or clinical interviews and mapped to their corresponding ICD-10 codes. The analysis will focus on the total number of coexisting chronic conditions per participant (disease burden) and the prevalence of major ICD-10 diagnostic chapters (e.g., cardiovascular, metabolic, musculoskeletal diseases).

Assessment of the RNA profile isolated from total plasma samples using next-generation sequencing (NGS) to evaluate changes over time.

Assessment of the RNA profile isolated from purified extracellular vesicles using next-generation sequencing (NGS) to evaluate changes over time.