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"Thinking on future"

Here I am going to explain the complete methodology we plan to get the specific objectives of the global project. 

The main objectives are the following:

1. To assess for the first-time levels of physical activity, sedentary behaviors and sedentary breaks in a representative sample of non-institutionalized older female and male adults using accelerometers, providing prevalence of active, non-active and sedentary seniors according to the new guidelines on PA and sedentary patterns.

2. To update gender and age-specific physical fitness normative values in non-institutionalized Spanish older adults with special focus on absolute and relative muscle power.

3. To provide an updated prevalence of female and male overweight, obesity, sarcopenia and sarcopenic obesity in a representative sample of the non-institutionalized Spanish elderly population.

4. To describe the changes in physical fitness, body composition, quality of life, vitamin D, biochemical blood parameters, cognitive status and frailty during a 12-year follow-up in a large sample of Spanish adults aged 65 or over.

5. To ascertain whether the identified changes in physical fitness, body composition vitamin D, biochemical blood parameters, as well as the PA /sedentary patterns are associated with mortality in this population.

6. To analyze the independent and combined association of lifestyle behaviors (PA, sedentary, sleep and nutrition), physical fitness and cellular hallmarks of aging (BDNF, klotho protein) on cognitive performance and considering the moderation role of being APOE 𝜀4 allele carriers or non-carrier.

7. To analyze the independent and combined association of lifestyle behaviors (PA, sedentary, sleep and nutrition), physical fitness and cellular hallmarks of aging (BDNF, klotho protein) on frailty and considering the moderation role of APOE genotype.

8. To explore the independent and combined role of lifestyle behaviors (physical activity, sedentary and sleep patterns) and physical fitness as determinants of BDNF and klotho protein, and the moderation role of APOE genotype.

9. To examine the positive effect of a training program on single task, dual-task and cognitive performance in female and male olders, as an alternative to maintain their health related to quality of life.

10. To observe the benefits of an intervention program on reducing the number of falls in elders especially in women, as one way to improve their independence.

11. To investigate the effect of a training program on the improvement of biomarkers of vascular homeostasis, cellular aging and cognitive deterioration levels and quality of life, and its relationships with cognitive function, physical fitness and falls in elders.


METHODOLOGY for a new comprehensive project:


1. National cross-sectional and longitudinal project (subproject 1)

This study includes participants from all the nodes participating in the coordinated project and the specific PA and sedentarism cut-off validation sub-study.


1.1. Sample and design of the study

Until now, EXERNET-elder projects were composed by one cohort (called original cohort). In this new project, in order to update reference values of physical fitness and body composition and establish new references for PA and sedentarism in Spanish older adults, the historic cohort will turn into an open cohort. New subjects will be recruited according to the same sampling method and following the same inclusion and exclusion criteria as the former ones.

This study will be carried out in 5 Spanish regions: Aragón, Castilla-La Mancha, Madrid, Extremadura and Andalucía. The aim is to recruit a representative sample of non-institutionalized Spanish seniors aged 65–92 years from social, civic and sport centers. Subjects will be selected by means of a multistep, simple random sampling, considering, first, the locations (five different regions from Spain) that ensure the geographical and cultural diversity of the sample, then three different cities of each region (the capital of the region and two other cities; one of 10 000–30 000 habitants and other of 30 000–100 000 habitants) and, finally, by random assignment of the civic and sports centers. For an estimated error of ±1.5%, and a variability P = q = 0.5, the established number of subjects is 3000 in order to guarantee a representative sample of the whole country. The total number of subjects will be uniformly distributed in the five regions (n= 600) and in their corresponding cities. Within the Regions, socio-geographical position of the cities will be also considered.  The exclusion criteria will be people under 65 years; those suffering from cancer and/or dementia; and those who are living in nursing homes and/or are not independent or able to take care of themselves.

Four of the regions already participated in the first, second and third evaluation of the EXERNET study. From those who participated at baseline, 3136 subjects, a total of 740 agreed to participate and finally 651 completed all measurements from the last follow up (EXERNET-Elder 3.0 project). This means a survivor rate of 23.6%. For this project, first, all survivors will be contacted again and encouraged to participate in the study. According to the response rate, the rest of the subjects needed to complete until 600 in each of the four regions will be calculated. Those who started in 2008 aged 65 + will be aged 78 + in 2021. To have an equal age distribution, for this project, mainly subjects aged <78 years will be recruited. For example, if 150 former subjects in each region agree to participate, 450 subjects will be newly recruited according to the above-described sampling method. Sex distribution will also be considered, to have an adequate female/male sample.

The region of Andalucía is a new cluster in the EXERNET study. Sampling method will follow the same procedure. Age and sex distribution will follow the same structure as the rest of regions. Following the same example, 150 subjects will be aged > 78 years, and the rest uniformly and randomly distributed among subjects 65 to 78 years.

For blood analysis in the coordinated project, a subsample of 1000 will be recruited. They will be randomly selected from the whole sample, assuming an error of 3%.

Data management

EXERNET-Elder 4.0 data and database will be managed centrally. Researchers from UPM will be responsible for the EXERNET database. The researcher in charge of the data and responsible for the project will be Prof. Marcela González-Gross (MGG). Personal data will be anonymized in order that no other researcher will have access. Paper-based personal data will be sent to UPM, where they will be stored in a secure cupboard, which has been specifically bought for this commitment. There will be a need to have access to personal data from the subjects who have already participated in the former EXERNET studies. Names and telephone numbers will be available for a group of 3 researchers (one in Zaragoza, one in Madrid and one in Toledo), who under the supervision of MGG will contact the subjects. All subjects participating in the study will get a unique code by a random lottery. There will be more codes available than subjects.

As UPM is part of the REDCap consortium, data acquisition during the field work, mainly questionnaires, will be preferably obtained online by means of REDCap, which is a secure web application for building and managing online surveys and databases. While REDCap can be used to collect virtually any type of data in any environment (including compliance with 21 CFR Part 11, FISMA, HIPAA, and GDPR), it is specifically geared to support online and offline data capture for research studies and operations. The rest of data will be also stored in REDCap. Data introduced manually will be typed twice by two different researchers in order to minimize typing errors. Apart from MGG, a group of 3 researchers (one in Zaragoza, one in Madrid and one in Toledo) will have access to REDCap. At UPM, REDCap is allocated at CESVIMA, a highly secure server from the university. Data analysis will be performed completely anonymized.

EXERNET-Elder 4.0 will comply with the Legislation related to data protection, particularly the European Union General Data Protection Regulation (EU 2016/679) and the Spanish Ley Orgánica 3/2018 de Protección de Datos y Garantías de los Derechos Digitales but also with it will be abided by the following regulations: The Charter of Fundamental Rights of the EU and Law 14/2007, 3 July, on biomedical research (BOE, 4 July 2007). All the study will be performed according to the Ethical Rules and Recommendations Guiding Physicians in Biomedical Research Involving Human Subjects of the Declaration of Helsinki.

The EXERNET cohort study had always the Research Ethics Committee of Aragón (CEICA) permission. The current project will be also authorized by the CEICA, the Committee in Extremadura and has also been submitted for evaluation to the Research Ethics Committee of Cádiz using the PEIBA platform ( 

Once the data collection phase of the study is finished, a report of the results will be made and delivered to each participant. It will consist of three parts: a body composition report (including bone mass in the nodes that perform this test), a physical fitness report, and a blood test report.


1.2. Accelerometry validation sub-study (objective 1)


Ninety subjects will be recruited through the Frailty Consultation of the Geriatric Department of the Virgen del Valle Hospital in Toledo. Criteria for inclusion/exclusion are detailed below:

Inclusion criteria: (1) Age ≥65 years old; (2) Able to walk independently; (3) SPPB score >3 points.

Exclusion criteria: (1) Currently undertaking a physical conditioning program; (2) A Mini Mental Scale score <18 points; (3) A marked walking disorder; (4) A clinically diagnosed illness contraindicated for PA.

Experimental protocol

Tests will be carried out at the Virgen del Valle Hospital in Toledo for two days. All following procedures will be conducted under qualified health professionals' supervision:

During the first day, the evaluation of the resting metabolic rate and physical function tests will be performed. The attendance conditions will be (1) Fasting, except for water, 7 hours before; (2) Refrained from smoking 3 hours before; (3) Refrained from caffeine 4 hours before; (4) Refrained from exercise of moderate intensity 12 hours before or vigorous intensity 24-48 hours before1.

- Evaluation of resting metabolic rate: A stationary Jaeger Oxycon Pro gas analyser (Erich Jaeger GmbH, Hoechberg, Germany) will be used to measure the resting metabolic rate. Participants must undergo a prior resting period of at least 20 minutes before the start of the measurement. After that, participants will undergo an additional 30-minute record period1. To achieve an accurate result, the 5-minute interval of the last 25 minutes with a coefficient of variation of less than 10% for O2, CO2 and VE and 5% for RER, will be selected2. The resting metabolic rate will be used to calculate the MET value for each participant.

- Evaluation of physical function: The Short Physical Performance Battery (SPPB) will be used to evaluate the functional status of the subjects3.

During the second day, the evaluation of physical activities will be carried out. Each subject will undergo a set of sedentary and walking stages while wearing the hip accelerometer and the gas analyzer.

- Physical activity stages: Stages were designed to simulate sedentary behaviors, light physical activities, and moderate-to-vigorous physical activities. Each stage will last for 5 minutes and will be divided into two blocks in an increasing sequence of intensity:

1. Sedentary activities: (1) Sitting in a chair; (2) Standing.

2. Light and moderate-to-vigorous PA (incremental protocol): Subjects will perform different walking stages on a treadmill (Lode B.V., Groningen, Netherlands) at a minimum speed of 0.5 km/h, gradually increasing by 0.3 km/h at each successive stage. The test will be completed when energy demand reaches 3 METs or more.

After the completion of each stage, sufficient rest time will be given to allow the physiological markers to return to the previous rest levels. Once the subject is considered recovered, next stage will start.

- Use of accelerometry: An ActiGraph wGT3X-BT and GT9X Link accelerometer (ActiGraph, Pensacola, FL, USA), located on the subject’s right hip and configured to measure at 60Hz, will be used.

- Evaluation of energy expenditure: The activity intensity will be classified by the METs system, categorizing each stage as sedentary activity (≤1.5 METs)4, light PA (>1.5-3 METs) or moderate-to-vigorous PA (>3 METs)5. Energy expenditure will be assessed using a stationary gas analyzer (Jaeger Oxycon Pro; Erich Jaeger GmbH, Hoechberg, Germany).

Statistical analysis

For result analysis, participants will be divided into three groups according to the SPPB score (SPPB ≥4 and ≤6 points: moderate dependence; SPPB ≥7 and ≤9 points: slight dependence; and SPPB ≥10 points: autonomous). For each activity, descriptive statistics concerning intensity measurements (METs) and accelerometry (counts) will be calculated.

ROC curves will be applied to determine sex-specific cut-off points (sedentary behaviour, light PA and moderate-to-vigorous PA)6,7. Points maximizing sensitivity and specificity (area under the curve) will be selected. A 95% confidence interval will be established for this test.

1.3. General measures of subproject 1. (This part is common for subprojects 1 and 2; objective 2-8)

As in our previous cross-sectional and longitudinal Exernet projects, in order to facilitate the process during the experimental phase and enable a functional data collection and the participants’ voluntary contribution to the project, each subject will carry out all the tests in two days. An identical order for all participants would be established in order to avoid possible alterations at the time of carrying out the tests (especially the physical tests) that could influence the results. The first day will be dedicated to the completion of questionnaires, fasting blood extraction and evaluation of the body composition and the second day to the performance of the physical fitness tests.

Participants of both, cross-sectional and longitudinal phases of this project will perform the following measurements:

Sex-specific physical fitness assessment

Fitness assessments will be based on the Senior Fitness Test8 and the Eurofit Battery9, in addition to other complementary tests to evaluate the following variables:

- Balance and agility: Static balance will be evaluated through the Flamingo test8, which measures the maximum time (s) the person is able to stand with one foot with arms on hips and with the contralateral foot leaning on the opposite ankle. Agility and dynamic balance will be assessed through three different tests: 2.45m Up-and-Go test8, Timed Up & Go Test (TUG)10 and TUG with a simultaneous cognitive task (TUGcog)9. They measure the time taken (s) to rise from a chair, turn over around a cone situated at 2.45 m (2.45m Up-and-Go) or 3 m (TUG and TUGcog), come back and sit. During TUGcog participants must name as many different animals or colours as possible out loud as they perform the test as quickly as possible.

- Flexibility: Lower limb flexibility will be evaluated through the Chair Sit-and-Reach test, which consists of measuring the distance (cm) from the tips of the middle fingers to the upper part of the shoe, testing one leg at a time. Upper limb flexibility will be tested by Back Scratch Test 8 in which it is measured the distance (cm) of overlap, or the distance between the tips of the middle fingers of both arms.

- Dynamic strength: Lower limb strength will be tested by Chair Stand Test, which measures the number of repetitions elders are able to stand up and sit down from a chair in 30 s. Upper limb strength will be tested by Arm Curl Test in which elders are required to lift a dumbbell of 4 kg (men) or 2.5 kg (women) the maximum number of repetitions within 30 s.

- Maximum isometric strength: Handgrip strength will be tested using a digital dynamometer (Takei TKK 5401, Tokyo, Japan) with a precision of 0.1 kg. When performing the test, participants are encouraged to apply the maximum strength possible.

- Aerobic endurance: Cardiorespiratory fitness was tested through the 6-minute Walk Test in which elders had to cover as much distance as possible in the 6-minute time limit around a rectangular course of 46 m. At least 2 participants must perform the test simultaneously.

- Walking speed: Maximum walking speed will be evaluated through the 30-m walk test49 that measures the time spent to walk 30 m as fast as possible, without running.

- Muscle Power: Muscle power will be assessed by the 30-s STS power test11–13 using a mobile application (PowerFrail, Spain). Briefly, the performance in the 30-s STS test will be video recorded and muscle power will be calculated using the participants’ body mass and height, chair height and the number of repetitions completed in 30 s. After the cue “ready, set, go!”, the subjects will perform STS repetitions as rapidly as possible on a standardized armless chair (0.45 m seat height) from the sitting position with their buttocks touching the chair to the full standing position, and with their arms crossed over the chest. Strong verbal encouragement will be given throughout the test. The participants will be familiarized with 2 attempts before the definitive STS test is performed. Then, the mobile application will calculate muscle power according to the following equation:

STS power= (Body mass·0.9·g·[Height·0.5-Chair height])/([Time/(n of reps)]·0.5)

where body mass is indicated in kg, body height and chair height in m, and time in s. This equation is based on several biomechanical principles that have been clarified elsewhere12,13. Briefly, 0.9 is a coefficient applied to body mass to calculate the body mass that is displaced during the STS task (whole body mass minus shank and feet mass), 0.5 is a coefficient applied to body height to calculate leg length, and 0.1 is a coefficient applied to total 5-rep STS time to calculate the average duration of the concentric phase of 1 STS repetition. Relative muscle power will be calculated as absolute STS power normalized to body mass to calculate relative STS power (W·kg-1), which denotes the ability to produce mechanical power per unit of body mass. Allometric STS power (W·m-2), a scaled measure of mechanical power, will be obtained by normalizing absolute STS power to height². Finally, specific STS power (W·kg-1) will be obtained from the ratio between absolute STS power and legs lean mass and indicates the ability to exert lower-limb mechanical power per unit of lower-limb muscle mass.


Body composition and Anthropometric measurements

- Height, weight, fat mass and muscle mass: Height will be measured with a portable stadiometer of 2.10 m and a 0.001 m error margin (SECA, Hamburg, Germany). A portable bioelectrical impedance analyzer with a 200 kg maximum capacity and a +/- 50 g error margin (TANITA BC 418-MA Tanita Corp., Tokyo, Japan) will be used to assess the body weight (kg) and to estimate the body total fat mass, the percentage of body fat and the fat free mass. Body mass index (BMI) will be calculated following the formula BMI=weight (kg)/height (m)².

- Anthropometrics: The circumferences of waist and hip will be registered according to the International Society for the Advancement of Kinanthropometry (ISAK) protocol14 with a Rosscraft Anthrotape (Rosscraft Innovations Inc, Vancouver, Canada).

- Bone parameters:

Bone mineral content and density (BMC and BMD): this will be measured in a subsample of 1200 participants from Aragon and Castilla la Mancha. A DXA scanner (Hologic Horizon A, Hologic Corp., Software version 12.4, Bedford, Massachusettes, USA) will be used to evaluate areal BMD (g/cm2) at the whole body, lumbar spine (mean L1-L4) and proximal region of the femur (total hip, femoral neck, Ward's triangle and trochanter).

Bone Mineral Density and strength indices: this will be measured in a subsample of 600 participants from Aragon.  Bone mineral density (BMD) of trabecular and cortical bone (mg/cm³), both in radius and tibia of non-dominant limbs, will be evaluated using Peripheral Quantitative Computed Tomography (PQCT) (XCT 2000 Peripheral QCT Scanner, Ortometrix, INC). The device provides information about cross-sectional areas related to bone size (area), mass (mineral content), apparent tissue density and geometry (spatial distribution of mass). Moreover, strength indices are calculated using a combined measurement of cross-sectional geometry and tissue density. Additionally, we will also measure bone marrow adipose tissue with pQCT which is inversely related to DXA-measured bone mineral in caucasian women 15.We hope to compare the three conditions (robust, frail and pre-frail older adults), and establish possible associations between the measured aging biomarkers (explained below) and bone marrow adipose tissue in order to establish possible pathways that could decrease marrow adipogenesis and increase osteoblast differentiation. We will also explore the effects that medications have on this differentiation, as this population is usually polymedicated and glucocorticoids have shown to enhance marrow adipogenesis at the expense of osteoblast differentiation16 . This could also be the case for other medications and further research is needed in order to improve bone health in these polymedicated older adults.

Physical Activity and Sedentary behaviors

PA and sedentary time and behaviors will be assessed by objective methods (accelerometry) and subjective tools (validated questionnaires).

- Accelerometry: Participants will wear a triaxial accelerometer ActiGraph GT9X Link on the hip to record physical activity and sedentary periods during 7 consecutive days including two weekend days. This device has been validated to measure PA6. Monitor outputs will be processed using ActiLife software (ActiGraph Corp., LLC).

- Physical Activity Scale for the Elderly (PASE): PASE is a 12-items questionnaire that combines information related to frequency and intensity on leisure, household and occupational activities performed in the last week17.    

- Elderly EXERNET PA Questionnaire (EEPAQ): EEPAQ includes three previously validated questions about the time spent sitting, walking and doing household activities per day18.

Cognitive Performance

Cognition will be measured by a set of tests, internationally well-known and validated in older adults. The total duration of the cognitive evaluation ranged from 15 to 20 min.

- MoCA (Montreal Cognitive Assessment): The MoCA was designed as a rapid screening instrument for mild cognitive dysfunction19,20. In the last years, it has shown that MoCA is known to be more precise than other cognitive tests such as Mini Mental State Examination (MMSE)21.  It assesses different cognitive domains: attention and concentration, executive functions, memory, language, visuoconstructional skills, conceptual thinking, calculations, and orientation. Time to administer the MoCA is approximately 10 minutes. The total possible score ranged from 0 to 30, and the highest score indicates better performance.

In order to test the comparability between MoCA and MMSE, subproject 2 will perform both tests.

- Stroop Color and Word Test (SCWT): The SCWT is a valid and widely applied for examining cognitive flexibility, selective attention, and cognitive inhibition22,23. This test is divided in three conditions, the first condition consists in read names of colors printed in black ink, the second condition was read the colors printed in an “X”, and the third condition consisted in naming the color of the ink instead of reading the word since colour-words are printed in an inconsistent color ink (for instance, the word “blue” is printed in red ink). All conditions contain 100 words, and the time limits to 45 seconds for each condition. Total number of correct words was registered for each condition indicating that higher number of correct words, better performance.

Due to the expertise and specialization on cognitive performance, and with the aim to deepen the Interaction of lifestyle behaviors, APOE genotype and cellular hallmarks of aging with cognitive performance and frailty (Objectives 6-8) Andalucía node will additionally include a specific battery for cognitive assessment (i.e. MMSE, RAVLT, CDT, COWAT, BNT, TMT and WASIII) as well as a complementary biomarkers (BNF and APOE genotype).

Cognitive performance battery assessment

Additionally to MoCA and SCWT questionnaires, that will be measured in the whole sample, in subproject 2 a complete neuropsychological test battery will be applied, including a set of tests, internationally well-known and validated in older adults. The tests aim to assess cognitive impairment, learning and verbal episodic memory, visuoconstructive and visuospatial skills, verbal and semantic fluency, visual confrontation naming, cognitive flexibility, attention, and inhibition Total testing time range from 60 to 75 min. Detailed information about the constructs to be measured and tests chosen can be found below.

- Mini Mental State Examination (MMSE). The MMSE is a valid test widely used to evaluate cognitive status24,25. The MMSE measures five domains of cognitive functions: i) orientation, ii) registration, iii) attention and calculation, iv)delayed recall, and v) language and construction. The test is divided in two parts, the first one requires verbal answers and the maximal punctuation is 21 while the second part assesses the skills for naming, following different verbal and written instructions, writing a spontaneous sentence and copying interlocking pentagons, and its maximal punctuation is 9. The total punctuation summing each domain is computed ranging from 0 to 30, and the highest scores indicate better performance.

- Rey Auditory Verbal Learning Test (RAVLT). The RAVLT is a valid test that assesses learning and verbal episodic memory26. The test consists of 5 learning trials where a list A of 15 words is read and the participant is asked to recall words from the initial repeated list (named as immediate recall). Then, a distractor list (list B) with other 15 words is provided, and the participant is asked to recall words only once. After that, list A is again provided and the examiner required to the participant to recall one more time the words. After 20 min, they were asked again for the list A (named as delayed recall). Finally, participants need to answer yes/no in 75-item recognition discrimination test where the 15 initial words were included. Retention was, therefore, calculated as the number of words correctly recalled at 20 min divided by the number of words correctly recalled at trial 5. The higher number of words recalled, the best the performance.

- Clock Drawing Test (CDT). The CDT is a valid cognitive test that is used to detect deterioration of the visuo-constructive and visuospatial skills27,28. The test consists of drawing a clock with the numbers on the circle, showing the clock faces into specific time (11:10 o’clock). They are not allowed to look at a watch or another clock for help. The total punctuation is the sum of the scores given to sphere, numbers and clock faces, ranging from 0 to 10 being the highest scores the best performance28.

- Controlled Oral Word Association Test (COWAT). The COWAT is a valid instrument designed mainly for assessing verbal and semantic fluency29,30. This test consists of spontaneous production of words beginning with a designated letter (P, M and R for the Spanish version) within a minute for each letter. They are not allowed to say names, numbers or words in different tenses or with the same root. For semantic fluency, they will be required to say words related to the topic given, i.e., animals. Total phonetic and semantic punctuation will be calculated summing all items independently for each punctuation (the higher the number of words given, the better the performance).

- Boston Naming Test (BNT). The BNT is a valid and widely used for assessing visual confrontation naming31. Particularly, we applied the short version of the BNT containing 15-items. Participants were shown line drawings of common objects one at a time and asked to name them orally, ranging from simple (high-frequency vocabulary, e.g. bed) to rare words (low-frequency vocabulary, e.g. abacus). Administration requires a spontaneous response within a 20 seconds period. If there is no response, two kinds of prompting cues (one semantic and one phonemic) were given. Total punctuation will be calculated by the sum of the total correct responses at the beginning (without cues) and after the semantic cues (the higher correct responses, the better performance).

- Trail Making Test (TMT). The TMT is a valid and widely used test to assess executive functioning32. The TMT consists of two parts, part A and part B. The part A is based on number sequencing and assesses visual-perceptual abilities and participants have to draw lines to link numbers from 1 to 25 in ascending order. The part B focus on number and letter switching evaluates cognitive flexibility and consists of drawing a line to link the numbers and the letters alternatively following in ascending order (e.g. 1-A-2-B-3-C). The completion time will be registered in seconds (the lower duration, the best performance).

- Wechsler Adult Intelligence Scale III (WAIS III). The WAIS-IIIis a test battery that has long been the gold standard for the evaluation of cognitive abilities33. Specifically, we only use the digit span to evaluate working memory34. Participants will be asked to recall a list of numbers in the order that they were given (forward or backwards). The test is finished when for the same item the participant fails two attempts. A total number of correct answers were registered for each condition.

Health-related Quality of Life

- Euroqol-5D Questionnaire (EQ-5D): EQ-5D is a questionnaire that includes 5 dimensions: mobility, self-care, main activity, social relationships, pain and mood. It also includes a health-reported scale ranging from 0 to 100, with 0 being the worst imaginable state of health and 100 being the best35.


Frailty status and functional capacity

According to the literature, frailty can be defined with different methods and criteria. The scale included in this project is the most widespread in the scientific literature: the frailty phenotype of Fried. This test is based on five items: unintentional weight loss (more than 4.5 kg in the year before or at least 5% of body weight), self-reported exhaustion (felt especially tired during the last week), weakness (low grip strength), slow usual gait speed (4.5 m) and low physical activity (less than 2 h walking per week). When three or more of these items are met, the degree of frailty was reached, while only one or two items denoted pre-frailty36.

- Evaluation of physical function: The Short Physical Performance Battery (SPPB) will be used to evaluate the functional status of the subjects3.


Clinical and Analytical biological parameters (this will be measured in a subsample of 1000 participants)

- Blood collection and biochemical and molecular analysis: Venous blood sampling will be performed according to the recommendations of the European Federation of Clinical Chemistry and Laboratory Medicine Working Group for Preanalytical Phase and Latin American Working Group for Preanalytical Phase37. The blood collection will be performed through venous puncture on the antecubital vein using a 21 gauge butterfly needle (BD Vacutainer Safety-Lok™, BD Biosciences, Australia) into four different 4 mL Vacutainer® tubes (BD Biosciences, Australia), one containing citrate, and three containing EDTA and three total blood tubes.

Blood samples for biochemical analysis will be allowed to clot for at least 60 m, centrifuge, and then, serum will be collected. The biochemical analysis from this serum and hematologic analysis will be analyzed within 24 h after collection. We will collect blood samples from the antecubital vein after 12 hours of fasting. All participants will be requested to abstain from drugs and/or caffeine, to eat a standardized dinner before sampling, and to avoid any physical activity of moderate intensity (24 hours before) and/or vigorous intensity (48 hours before).

- Ageing biomarkers: Proposed proteins as aging markers will be measured in patients serum using a western blot with an internal calibration curve that allows a semi-quantitative approximation. The proteins of interest, S-Klotho, PEDF, Interleukin-1 and smp30 will be detected by using specific antibodies and standards with purified protein or synthetic peptides according to each case. The total protein content present in the samples will be quantified by measuring the absorbance at 280 nm. Protein separation will be carried out by electrophoresis on polyacrylamide gels under denaturing conditions (SDS-PAGE). For this, 12% acrylamide: bis-acrylamide gels (ratio 29: 1) will be used, with the BoltTM Mini Gel Tank electrophoresis kit (Thermo Fisher Scientific) operating at 100 V for 1 hour at room temperature. For the transfer of proteins to nitrocellulose membranes we will use the iBlotTM 2 dry transfer equipment (Thermo Fisher Scientific) (8 min program consisting of a first step of 4 min at 10 V and another of 4 min at 15 V). Then, the membranes will be blocked and incubated with specific primaries in each case. Finally, the membranes already incubated with HRP conjugated with secondary antibody, will be developed using the ImageQuant LAS 500 chemiluminescence CCD camera (GE Healthcare) and exposure to the chemiluminescence substrate for 3 min. Both the calibration curve and the sample membranes will be incubated and developed simultaneously. For the quantification of the bands obtained by this technique, a standard curve of known concentrations of specific peptides will be used that allows extrapolating the protein concentration as a function of the intensity obtained by Western Blot (technology in patent process, with a positive patentability report from the National Patent Office). In order to quantify the intensity of the bands obtained by Western Blot, the ImageJ v.1.50 software will be used and the values ​​and graphs will be processed using the OriginPro 8 software. The statistical analysis will be performed using the non-parametric Mann-Whitney U test.

- Vitamin D: Additionally, vitD [25(OH)D] will be analyzed in plasma through the ELISA kits (VITROS 25-OH Vitamin D Total Reagent Pack and VITROS 25-OH Vitamin D Total Calibrators), using the immunodiagnostic system VITROS 3600 and VITROS 5600 Integrated System with Intellicheck® technology (UK). The VITROS 25-OH Vitamin D Total Reagent Pack Kit is an enzymatic immunoassay that allows for a quantitative determination in blood serum or plasma, and it has been widely used for the study of vitD deficiency in the clinical setting. The sensitivity of this method is of 5nmol/L 25(OH)D, with a coefficient of variation under 1% and a prevalence of false positive and false negative cases lower than 5%.

BDNF and APOE genotype

Additionally to other blood parameters that will be measured in the whole sample, in the Andalucia sample (n = 600) the BDNF levels and APOE genotype will be determined.

- BDNF levels (Brain derived neurotrophic factor) will be measured in serum using the RAyBio Human BDNF (RAFER) ELISA Kit (Enzyme-linkerd immunosorbent assay).

- Apoliprotein E genotyping. DNA extractions will be performed for the genotyping of the specific polymorphisms of Apolipoprotein E. APOE genotyping will be performed using Applied Bio-systems TaqMan® SNP Genotyping Assay, C_3084793_20 and C_904973_10 corresponding to APOE SNPs rs429358 and rs7412, respectively, and run with ABI 7900HT Fast Real-Time PCR system. Diplotypes corresponding to APOE E3/E3, APOE E3/E4 and APOE E4/E4 will be identified.


Blood pressure and arterial stiffness

- Blood pressure: will be obtained by the ankle-arm index (AAI) method, which requires a conventional sphygmomanometer (Omron, HEM-7112), a manual continuous Doppler probe (Hadeco Bidop ES-100V3, Düsseldorf, Germany) and a small amount of conductive gel. AAI of each of two extremities will be obtained by the following formula: AAI = maximum systolic BP in the ankle or foot/maximum systolic BP in the arm. Before measurements, elders will be asked to bare four limbs and take a 5 min rest in a supine position.

- Peripheral vascular function (to be measured in the subsample with blood sampling -1000 participants-): Arterial stiffness will be assessed by pulse wave velocity analysis (carotid-femoral) using a state-of-the-art SphygmoCor system (AtCor Medical, West Ryde, SW, Australia). It uses a high-quality tonometer and an EKG to analyze the carotid and femoral pulse wave. The transition time will be measured according to measurement standards after a 10-minute supine rest in a quiet and quiet room38 .The speed of the pulse wave will be determined by adjusting the time for the distance between the carotid and the femoral artery. Subjects should abstain from food for the previous 60 minutes as well as coffee or another beverage with caffeine or other stimulants. Those subjects who reach P97 in wave speed, according to European standards of their age group and sex, will be classified as EVA (Early Vascular Aging).


Nutritional habits

Adherence to Mediterranean dietary pattern will be analyzed using a brief 14-item tool validated in the older Spanish population39,40. Each question scored 0 or 1, thus, questionnaire values range from 0 to 14. The higher the score is, the higher the adherence to the Mediterranean dietary pattern. Moreover, these scores will be used for grouping participants into 3 and 5 categories, similarly to previous studies in older adults41,42, establishing adherence levels to Mediterranean diet from low to high.

Survival data

All-cause mortality during the 12-years follow-up will be collected by consulting the Spanish National Death Index.


1.4. Exercise intervention study subproject 2 (Objectives 9-11)

1.4.1. The design and samples for the intervention study.

Subjects from the autonomous community of Extremadura will be involved in an intervention training program to evaluate the benefits on cognitive function, falls, SMP30, physical fitness, body composition and quality of life.

Sample size (RCT).

Based on the fall risk test and accepting an alpha risk of 0.05 and a beta risk of 0.1 in a two-sided test, 33 subjects are necessary for the experimental group and 33 in the control group to recognize a statistically significant difference greater than or equal to 2.1 units. The common standard deviation is assumed to be 2.45 and the correlation coefficient between the initial and final measurement is 0.55. A drop-out rate of 20% has been anticipated.

Intervention training program

The design and characteristics of the intervention are based on previous studies carried out in older adults43–46. According to the ACSM a moderate-intensity (%HRR 40–60%) physical exercise is an effective activity to improve physical fitness and health in adults, including elders47. The square-stepping exercise (SSE) is an exercise that has been shown to meet this intensity criterion47. The SSE program, performed during 40 minutes twice a week over 3 months, has been effective in reducing fall risk factor in older populations45,46. However, no studies analyze the impact of SSE on mental executive function and regucalcin and other biomarkers levels, as determinants of fall risk in the elderly.

On the other hand, some studies, a few of them in our research group, have analyzed the effect of dual-task (DT) in elderly48–50 or women with Fibromyalgia39. We found that the walking pattern is affected when an additional cognitive task is added to the primary motor activity, so the walking pattern can be safer and stable if the attention is focused on 2 simultaneous tasks48,49. There are no studies combining SSE and DT. Therefore, we are going to implement an SSE program combined with cognitive tasks. 

The SSE is performed on a thin felt mat (250 × 100cm) divided into 40 squares (25 × 25cm). Subjects walk (step by step) from one end of the mat to another end according to the step pattern provided. The SSE patterns include backward, forward, oblique and lateral steps, the complexity of the step patterns increase progressively. The SSE has 196 different step patterns developed in 3 levels and 8 categories, the progressively increasing levels go from Elementary, 1-2 categories, to Intermediate (1-3 categories) and finish in Advanced level (1-3 categories)

Therefore, the SSE intervention will be conducted during 6 months, 2 days per week, with a progressive increase in volume and intensity, all training sessions will have around 10 minutes of warm-up, then 40 minutes of SSE training and will finish with 10 minutes of cool down.

All tests that will be performed before (baseline) and after (6 months) of the SSE-DT intervention are descriptive in the coordinator project. To analyze the durability of the changes by the intervention, subjects will be measured 3 months after the end of the SSE-DT intervention.

Training periodization

- Experimental group: The experimental group will be performing the SSE-DT training, 2 times per week, for 6 months. The training will be conducted in groups of approximately 6 people at the time in the Faculty of Sport Science in Cáceres. The SSE protocol will start with an easier forward walking pattern to more complex pattern, backward, oblique and lateral steps. The difficulty will be increasing during the first 4 months, then the last two months (5º and 6ª) will be performed at maximum intensity in the SSE plus DT. The characteristics of the intervention are displayed in table 1.


Table 1. SSE-DT training program

Months            Weeks            Days per week  Duration of the SSE                 Levels             Categories       Cognitive task

1                        1--2                2                                   40 minutes               Elementary               1                   

                          2--4                2                                   40 minutes               Elementary               2                    x

2                        1--2                2                                   40 minutes               Intermediate             1                   

                          2--4                2                                   40 minutes               Intermediate             2                   

3                        1--2                2                                   40 minutes               Intermediate             3                    x

                          2--4                2                                   40 minutes                Advanced                1                   

4                        1--2                2                                   40 minutes                Advanced                2                   

                          2--4                2                                   40 minutes                Advanced                3                   

5                        1--2                2                                   40 minutes                Advanced                3                    x

                          2--4                2                                   40 minutes                Advanced                3                    x

6                        1--2                2                                   40 minutes                Advanced                3                    x

                          2--4                2                                   40 minutes                Advanced                3                    x

SSE: Square-stepping exercise; DT: Dual-task


Specific measurements of sub-project 2

- Executive function: In the framework of measuring balance-related tests (Biodex Balance, 10m-walking, …), subjects will perform the tests in single and dual tasks wearing nir-HEG headband to compare the brain activation.

Model X-Wiz to Cortical Activation Measures in Fp1 and Fpz/Cz Regions by Nir-HEG/Quantified-EEG/HRV System measurements. We will measure based on the headband and pendant wearable multimodal system developed to simultaneously combine the monitoring of HemoEncephalograpy in the frontal lobe (HEG), 2-channel Electroencephalography (EEG) by theta/Beta ratio, and heart rate (HRV) by finger sensor51.

- Falls risk, Fear of Falling: The falls risk will be assessed with the Biodex Balance System (BBS) (Biodex, Shirley, NY, USA). The clinical test of sensory integration of balance (CTSIB) will be performed in four conditions: firm surface with eyes open or closed and unstable surface with eyes open or closed. In all four tests, subjects have to maintain their feet on the platform of the BBS for 30 s with 10 s recovery between tests. The BBS is a circular platform that moves on anterior-posterior and medial-lateral axes simultaneously, so the participants have to control their balance and movement degree. The BBS interfaces with software Biodex, 1.08, (Biodex, Inc., Shirley, NY, USA) allowing the degree in each axis and then calculating the sway score. The sway index was used for statistical analysis.

Beside participants will be asked a single question about the number of falls that they have had in the last six months, and another one about the number of falls in the last year, additionally they will say their fear of falling (0 = no fear, to 100 = maximum fear) with a Visual Analog Scale (VAS).

1.5. General statistical analyses (coordinated project)

All statistical analysis will be performed with the statistical packages using common codes SPSS, Stata or R. Statistical significance will be established at 5%. Results from quantitative analyses will be presented as mean ± standard deviation while those about qualitative variables will be presented as the percentage of each category.  Normal distribution of the quantitative studied variables will be explored with Kolmogorov–Smirnov tests. Associations between variables will be studied by bivariate Pearson correlations and regression analyses.

To show the evolution of the recorded variables (longitudinal changes), analysis of variance (ANOVA) for repeated measures will be used. When necessary, appropriated covariates will be included in the model. Logistic and linear mixed models will also be used when appropriate. T-sample test for independent samples, ANOVA and ANCOVA, will be performed to study differences between groups.


To analyze associations between independent and dependent variables mathematical models using logistic regression analysis will be performed. In this case, results will be presented as Odds Ratio (expβ) and confidence interval (IC) set at 95%. Cluster analysis is planned to identify profiles of older people who, based on their gender, age, level of physical activity, physical condition, body composition and vitD status, may have a higher risk of frailty or mortality.


The methodological viability of the tasks and activities described in this section is high, as all measurements have already been performed in previous studies. Therefore, no insurmountable difficulties are expected in the execution of this coordinated project that would jeopardize the achievement of the objectives.



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