The CALERIE™ Legacy Study

Biological age will be quantified by the Klemera-Doubal Method (KDM), an algorithm-based measure. Biological age is expressed in years.

Healthspan will be assessed by sex-specific metabolic syndrome z-score (MS z-score). MS z-score was developed using high density lipoprotein cholesterol (HDL), triglycerides (TG), waist circumference (WC), fasting blood glucose (FBG), and mean arterial pressure (MAP = [systolic blood pressure + 2 x diastolic blood pressure]/3). SDs for HDL and WC in the equations are sex specific.

Women: MS z-score = [45-HDL]/SDHDLW+ [TG-150]/SDTG + [WC-88]/SDWCW + [FBG-100]/SDFBG + [MAP -100]/SDMAP

Men: MS z-score = [40-HDL]/SDHDLM+ [TG-150]/SDTG + [WC-102]/SDWCM + [FBG-100]/SDFBG + [MAP -100]/SDMAP

W = women M = men

This score does not have a unit.

Biological age will be quantified by homeostatic dysregulation, an algorithm-based measure. Biological age is expressed in years.

Biological age will be quantified by Levine Phenotypic Age, an algorithm-based measure. Biological age is expressed in years.

Height will be measured in cm.

Weight will be measured in kg.

Weight circumference will be measured in cm.

Hip circumference will be measured in cm.

BMI (kg/m2) will be calculated using height and weight measurements.

Systolic and diastolic blood pressure will be measured in mmHg.

Pulse rate will be measured in beats/min.

Respiration rate will be measured in breaths/min.

Whole-body fat mass (kg, %) as measured by dual-energy X-ray absorptiometry.

Whole-body fat-free mass (kg, %) as measured by dual-energy X-ray absorptiometry.

Whole-body bone mineral content (g) as measured by dual-energy X-ray absorptiometry.

RMR will be measured in kcal/d by indirect calorimetry using a ventilated hood system.

RQ will be measured by indirect calorimetry using a ventilated hood system. This is a ratio (V̇CO2/V̇O2) and does not have a unit.

Maximal aerobic capacity (V̇O2max) will be measured in mL/kg/min using the Cornell incremental treadmill test.

IL-6, a blood-based biomarker of aging proposed by the Targeting Aging with MEtformin (TAME) Biomarkers Workgroup, will be measured in pg/mL.

Insulin, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in μIU/mL.

Insulin (μIU/mL) will be measured at 0 minutes (pre-dose) during an oral glucose tolerance test (OGTT) to evaluate glucoregulatory response.

Insulin (μIU/mL) will be measured at 30 minutes post dose during an OGTT to evaluate glucoregulatory response.

Insulin (μIU/mL) will be measured at 60 minutes post dose during an OGTT to evaluate glucoregulatory response.

Insulin (μIU/mL) will be measured at 90 minutes post dose during an OGTT to evaluate glucoregulatory response.

Insulin (μIU/mL) will be measured at 120 minutes post dose during an OGTT to evaluate glucoregulatory response.

IGF-1, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in ng/mL.

HbA1c, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in mmol/mol.

TNFRII, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in ng/mL.

hsCRP, a blood-based biomarker of inflammation, will be measured in μg/mL.

GDF15, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in pg/mL.

Cystatin C, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in mg/L.

NT-proBNP, a blood-based biomarker of aging proposed by the TAME Biomarkers Workgroup, will be measured in pg/mL.

SASP proteins, blood-based biomarkers of cellular senescence, will be measured using a SASP panel.

Urinary iPF(2α)-III, a biomarker of systemic oxidative stress, will be measured in ng/mL.

Urinary 2,3-dinor-iPF(2α)-III, a biomarker of systemic oxidative stress, will be measured in ng/mL.

Urinary iPF(2α)-VI, a biomarker of systemic oxidative stress, will be measured in ng/mL.

Urinary 8,12-iso-iPF(2α)-VI, a biomarker of systemic oxidative stress, will be measured in ng/mL.

Creatinine, a byproduct of routine activity in the muscle, will be measured in urine in mg/mL.

DHEA, a blood-based steroid hormone, will be measured in μg/dL.

IL-1b, a blood-based marker of inflammation, will be measured in pg/mL.

IL-8, a blood-based marker of inflammation, will be measured in pg/mL.

TNF-α, a blood-based marker of inflammation, will be measured in pg/mL.

IGFBP-1, a blood-based transport protein, will be measured in ng/mL.

TGFB1, a blood-based marker of immune function, will be measured in ng/mL.

IGFBP-3, a blood-based transport protein, will be measured in ng/mL.

PDGF-AB, a blood-based marker of cellular function, will be measured in ng/mL.

ICAM-1, a blood-based marker of inflammation, will be measured in ng/mL.

Growth hormone, a blood-based bio-marker of cellular function, will be measured in ng/mL.

MCP1, a blood-based marker of inflammation, will be measured in pg/mL.

Leptin, a hormonal regulator of energy balance, will be measured in pg/mL.

Total adiponectin, a protein hormone that regulates several metabolic processes, including glucose regulation and fatty acid oxidation, will be measured in μg/mL.

High molecular weight adiponectin, a protein hormone that regulates several metabolic processes, including glucose regulation and fatty acid oxidation, will be measured in μg/mL.

Knee extension at 60 degrees per second will be measured by isokinetic dynamometry and expressed in Nm.

Knee extension at 180 degrees per second will be measured by isokinetic dynamometry and expressed in Nm.

Knee flexion at 60 degrees per second will be measured by isokinetic dynamometry and expressed in Nm.

Knee flexion at 180 degrees per second will be measured by isokinetic dynamometry and expressed in Nm.

Handgrip strength will be measured via a hand dynamometer and expressed in kg.

Physical functioning as measured by a physical performance battery. This physical performance battery provides a composite score ranging from 0 to 12 and includes results from the balance, gait and chair stand tests.

Cognition as measured by the Cambridge Neuropsychological Test Automated Battery (CANTAB). The CANTAB evaluates six main areas of cognitive function: reaction time; verbal recognition memory; intra-extra dimensional set shift; rapid visual information processing; delayed matching to sample; and spatial working memory.

Dietary intake in calories (kcals/day), as measured by multiple-pass 24-hour dietary recalls and Nutrition Data System for Research (NDSR) software.

Dietary carbohydrates (grams and % of energy intake) will be measured by multiple-pass 24-hour dietary recalls and Nutrition Data System for Research (NDSR) software.

Dietary proteins (grams and % of energy intake) will be measured by multiple-pass 24-hour dietary recalls and Nutrition Data System for Research (NDSR) software.

Quality of life as measured by the RAND 36-Item Short Form Survey (SF-36). The RAND SF-36 measures eight aspects of health: physical functioning, role limitations due to physical health problems, role limitations due to personal or emotional problems, energy/fatigue, emotional well-being, social functioning, bodily pain, and general health perceptions. It also includes a single item that provides an indication of perceived change in health. Each item is on a 3- or 5-point Likert scale (1-3 or 1-5). Each response is transformed to a recoded value of 0-100, and the transformed scores of the 9 subscales are averaged to derive a composite score. Higher scores indicate more favorable health states.

Depression status as measured by the Beck Depression Inventory II (BDI-II). The 21-item BDI-II measures depression severity. The score range is 0-63, with higher scores indicating greater depression severity. Nineteen items are on a 4-point Likert scale (0-3). Two items are on a 7-point Likert scale, and responses are scaled to 0-3. The values are summed to calculate the total score.

Mood as measured by the Profile of Mood States Second Edition (POMS 2). The POMS 2 assesses six mood subscales using 65 items, each on a 5-point Likert scale (0-4): tension-anxiety (9 items, score range 0-36), depression-dejection (15 items, range 0-60), anger-hostility (12 items, range 0-48), vigor-activity (8 items, range 0-32), fatigue-inertia (7 items, range 0-28), and confusion-bewilderment (7 items, range 0-28). High vigor-activity scores reflect a good mood or emotion, and low scores in the other subscales reflect a good mood or emotion. Total mood disturbance, which is calculated by subtracting the vigor subscale score from the total subscale score (range 0-200), also will be assessed.

Sleep as measured by the Pittsburgh Sleep Quality Index (PSQI). The PSQI measures sleep quality and disturbance over the course of one month using seven subscales: subjective sleep quality; sleep latency; sleep duration; habitual sleep efficiency; sleep disturbances; use of sleeping medication; and daytime dysfunction. The scoring range of each subscale is 0-3. The scoring range of the Global PSQI (the composite score of the seven subscales) is 0-21. For both the subscales and Global PSQI, higher scores indicate greater difficulties with sleeping.

Sexual function as measured by the Derogatis Interview for Sexual Functioning - Self Report (DISF-SR). Both versions of the DISF-SR (male and female) are scored from 20 to 75, with higher scores indicating higher sexual functioning.

Food cravings as measured by the Food Cravings Questionnaire-Trait (FCQ-T). The FCQ-T measures frequency and intensity of food cravings. It has 39 items on a 6-point Likert scale (1-6) and a score range of 39-234. Higher scores indicate more frequent and intense food cravings. Scores on all items are summed for a total score.

Dietary restraint, disinhibition, and hunger as measured by the Three-Factor Eating Questionnaire (TFEQ). The 51-item TFEQ will be used to measure dietary restraint (21 items), disinhibition (16 items), and hunger (14 items). Item responses are scored as 0 or 1 and summed. Higher scores indicate higher levels of restrained eating (range 0-21), disinhibited eating (range 0-16), and predisposition to hunger (range 0-14).

Disordered eating as measured by the Multiaxial Assessment of Eating Disorder Symptoms (MAEDS). MAEDS will be used to assess six domains of disordered eating: binge eating (range 8-56), restrictive eating (range 9-63), purgative behavior (range 7-49), fear of fatness (range 11-77), avoidance of forbidden foods (range 10-70), and depression (range 11-77).

Body shape perceptions as measured by the Body Shape Questionnaire (BSQ). Items on the BSQ are rated on a 6-point Likert-type scale. The score ranges from 34-204 points. Higher scores indicate increased concern regarding body shape.

Physical activity as measured by the 7-Day Stanford Physical Activity Recall (PAR). The PAR estimates an individual's time spent in sleep and physical activity for the 7 days prior to the interview. Energy expenditure based on time spent in sleep and physical activities can be estimated. Sub-scores of energy expenditure for each of the following activities are calculated, summed, and averaged to estimate energy expenditure as kilocalories per day: sleep, moderate physical activity, hard physical activity, and very hard physical activity. Higher scores indicate greater physical activity per day. Metabolic equivalent (MET) value for each activity is provided: 1 MET for sleep, 4 METs for moderate activity, 6 METs for hard activity, and 10 METs for very hard activity. Time spent in each activity over 7 days is multiplied by the appropriate MET value. The minimum value for each activity is zero; there is no maximum value.

Resilience as measured by the 25-item Connor-Davidson Resilience Scale (CD-RISC-25). CD-RISC-25 is scored from 0 to 100, with higher scores indicating higher resilience.

Total cholesterol will be measured in mg/dL using a standard lipid panel.

HDL-C will be measured in mg/dL using a standard lipid panel.

Triglycerides will be measured in mg/dL using a standard lipid panel.

LDL-C will be calculated in mg/dL using measured values of total cholesterol, HDL-C, and triglycerides.

Glucose (mg/dL) will be measured at 0 minutes (pre-dose) during an OGTT to evaluate glucoregulatory response.

Glucose (mg/dL) will be measured at 30 minutes post dose during an OGTT to evaluate glucoregulatory response.

Glucose (mg/dL) will be measured at 60 minutes post dose during an OGTT to evaluate glucoregulatory response.

Glucose (mg/dL) will be measured at 90 minutes post dose during an OGTT to evaluate glucoregulatory response.

Glucose (mg/dL) will be measured at 120 minutes post dose during an OGTT to evaluate glucoregulatory response.

Insulin resistance will be calculated using the Homeostatic Model Assessment (HOMA) and measured values of fasting glucose (mmol/L) and fasting insulin (μ/L): (fasting glucose × fasting insulin) ∕ 22.5

Beta-cell function will be calculated using HOMA-β and measures of fasting insulin (μ/L) and fasting glucose (mg/dL): HOMA-β (%) = (360 × fasting insulin) ∕ (fasting glucose - 63)

Insulinogenic index will be calculated as the ratio of change in plasma insulin (I) from 0 to 30 min of the OGTT to the change in plasma glucose (G) over the same period (∆I0-30 min ∕ ∆G0-30 min).

Insulin sensitivity index will be calculated as 1 ∕ fasting insulin (μ/L).

The Oral Disposition Index will be calculated as the product of insulinogenic index and insulin sensitivity index.

T3, a blood-based marker of thyroid function, will be measured in ng/dL.

T4, a blood-based marker of thyroid function, will be measured in ng/dL.

TSH, a blood-based marker of thyroid function, will be measured in μIU/mL.

ALKP, a blood-based marker of liver function, will be measured in IU/L.

ALT, a blood-based marker of liver function, will be measured in U/L.

AST, a blood-based marker of liver function, will be measured in U/L.

Albumin, a blood-based marker of liver and kidney function, will be measured in g/dL.

BUN, a blood-based marker of kidney function, will be measured in mg/dL.

Total proteins are computed as a sum of albumin and globulin. Total proteins are indicative of blood vessel and immune system function and will be measured in g/dL.

Bilirubin, a blood-based marker of liver function, will be measured in mg/dL.

CPK, an enzyme marker of energy homeostasis, will be measured in U/L.

LDH, an enzyme marker of liver function, will be measured in U/L.

Globulin, a blood-based marker of liver function, will be measured in g/dL.

A/G ratio is a blood-based marker used to monitor nutritional status and immune system, kidney, and liver function. This marker is a ratio, and therefore does not have units.

Uric acid, a blood-based marker of purine metabolism, will be measured in mg/dL.

Calcium, a blood-based mineral, will be measured in mg/dL.

Phosphorus, a blood-based mineral, will be measured in mg/dL.

Magnesium, a blood-based mineral, will be measured in meq/L.

Sodium, an electrolyte that reflects the body's fluid balance, will be measured in meq/L.

Potassium, an electrolyte that reflects the body's fluid balance, will be measured in meq/L.

Chloride, an electrolyte that reflects the body's fluid balance, will be measured in meq/L.

Apoprotein A1, a blood-based marker of lipid metabolism, will be measured in mg/dL.

Apoprotein B, a blood-based marker of lipid metabolism, will be measured in mg/dL.

The oxidized (NAD⁺) and reduced form (NADH) of nicotinamide adenine dinucleotide will be measured in plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and/or fluorometric enzymatic cycling assays for NAD⁺/NADH. Concentrations will be reported as nmol per mg protein or micromolar (µM) in plasma.