Vitamin D Supplementation in Estonian Conscripts

The Influence of Vitamin D on the Physical Performance and General Health of Conscripts of the Estonian Defence Forces

A longitudinal, double-blinded, randomized, trial, with a 10 month follow-up period will be conducted between July 2021 to May 2022

I DETAILED DESCRIPTION OF THE SUBJECTS AND THEIR RECRUITMENT Four hundred volunteers from the 2nd Infantry Brigade of the Kuperjanov Infantry Battalion (Võru, Estonia) aged 18-30 are invited to participate in this study. To this end, a general meeting will be held at the beginning of the conscription. The participants will be given an overview of the study, its objectives and methods, all the risks that may be involved, and the potential benefits. The meeting will emphasise the voluntary nature of the study and the fact that non-participation in the study does not lead to changes in the overall cost of their conscription. Conscripts wishing to participate in the study can sign an information sheet and informed consent form to participate in the study after the meeting. Based on previous studies (experience of our working group in 2014-2017), the participation of about 150-170 conscripts is expected (25-30% of the total number). Places of the study: University of Tartu Clinic, Sports Medicine and Rehabilitation Clinic, Sports Traumatology Centre, Puusepa 1a, Tartu, 51014 Recruitment of subjects, general medical assessment and completion of questionnaires, blood sampling, physical tests, and bone densitometry analysis. Kuperjanov Infantry Battalion, 2nd Infantry Brigade (Kose tee 3a, 65603, Võru) Determination of vitamin D, PTH, and Ca in the blood: Synlab Eesti OÜ, Teguri 37b, 50107, Tartu The questionnaires used in the study will be completed on site at the Kuperjanov Infantry Battalion. The subjects are divided into two groups by a computer with double-blind randomisation in a 1 : 1 ratio - subjects receiving 4,000 IU of vitamin D or a 100 µg capsule (Group I, n = 75-85) and subjects receiving 600 IU or 15 µg vitamin D capsules (Group II, n = 75-85) of the same shape and colour once a day with breakfast The corresponding capsules are pre-prepared in agreement with the manufacturer (HC CLOVER Productos y Servicios, S.Lk) and packed in standard jars (100 capsules per jar; a total of three jars with the corresponding number of capsules for ten months should be used for the study). Each jar is coded individually for each subject in accordance with the randomised groups. Serum levels of vitamin D (25(OH)D), s-PTH, s-Ca, and s-iCa are determined in all subjects during the visits (SynLab Eesti OÜ, Tartu). Analyses are taken four times during the study (visits): July, October, February, and April/May. All analyses are determined at the end of the study at the same time to reduce intra-assay variability due to the use of different kits. For all health-related problems related to the study, as well as other problems related to the study, conscripts are consulted by the orthopaedist in charge (Dr Leho Rips). If there are any health problems not related to the study, the medical staff of Kuperjanov will inform the principal researcher (Dr Leho Rips). The subjects have the right to contact the principal researcher directly in case of problems related to the study. Exclusion from the study: - refusal of a conscript or withdrawal of consent, departure from military unit, force majeure, acute trauma, or illness in the course of conscript requiring hospital treatment. All these cases are recorded and taken into account in the subsequent analysis. II DETAILED DESCRIPTION OF THE STUDY METHODOLOGY Anthropometric measurements. The length of the body (LB) is measured with a metal anthropometer in a vertical position, the heels are together and the person measuring the subject ensures that the lower eyelid and the outer ear canal of the subject are in a horizontal plane. To determine body weight (W), the subject should wear as little clothes as possible. An electronic scale with a measuring accuracy of ± 0,1 kg is used for the measurement. The formula BMI = W / LB [kg/m2] is used to calculate the body mass index. Vitamin D in serum ((25(OH)D is determined by chemiluminescence immunoassay (CLIA). (LIAISON 25 OH Vitamin D TOTAL Assay (REF 310600, DiaSorin Inc, USA)) with a sensitivity of 4 ng/ml and an intra-assay variability coefficient (CV) of 0.1-3.8%. Vitamin D levels in the blood are determined four times during the study period to explain the effects of strain, Estonian climate, and supplementation on serum concentrations (at the beginning of the conscript, twice in the middle of the conscript, and in the final phase). Calcium (s-Ca), ionised calcium (s-iCa), and parathyroid hormone (s-PTH) are determined from blood serum by potentiometry and chemiluminescent immunoassay (CLIA), respectively. All blood tests are taken at the same time in the morning before breakfast and during the same periods, analogously to vitamin D. About 5 to 8 ml of blood is taken during each blood test, a total of 20 to 32 ml. This is about 2 tablespoons of blood throughout the study. Assessment of knee pain. The visual analogue scale VAS is used where '0' - no pain and '10' - maximum pain, associated with the KOOS questionnaire to assess subjective complaints related to the knee joint. Subjective assessment of knee joint function by the subject. The subjects evaluate the subjective complaints and function of the knee joint with a questionnaire (Knee Injury and Osteoarthritis Outcome Score, KOOS), which is used to find out their assessment of the condition of their knee(s) and how well they can cope with daily activities. It is possible to get a maximum of 100 points (no complaints) and a minimum of 0 points, which means extreme complaints. Hand grip strength is determined by isometric contraction while standing, upper limb away from the body, using a hand dynamometer (Lafayette Instrument Co., USA). The studies use maximal hand grip strength as an indirect indicator of whole-body muscle strength. Subjective assessment of physical activity by the subject. The Baecke Physical Activity Questionnaire consists of three parts and is based on a person's subjective assessment of their work, sport, and leisure (it is possible to get 1-5 points for each question). To interpret the results, the number of points in each subsection and the total number of points in the whole questionnaire are given. A similar questionnaire has been used to determine physical activity in previous studies in conscripts. IPAQ International Physical Activity Questionnaire, which assesses the level of physical activity in the last 7 days. The assessment of subjective pain in different regions of the body is done with the Cornell Musculoskeletal Discomfort Questionnaires (CMDQ), where the intensity of pain and the region of the body can be noted. Pain questionnaire ( to assess pain in different regions of the body on a scale from 0 (none) to 10 (maximum possible pain). The assessment of subjective health status and health-related quality of life by the subject is performed using standard questionnaires SF-36, RAND36, and BDI - Beck Depression Inventory. The questionnaires have been validated in Estonia and are valid for identifying health-related aspects. The questionnaires are based on the subject's assessment of their health aspects. The SF-36 questionnaire identifies the health status with a descriptive health profile and is more sensitive than other questionnaires. It also calculates the total physical and mental health scores. The BDI questionnaire is based on the assessment of the subject concerning aspects related to their mental health. Evaluation of emotional well-being and somatic symptom disorder symptoms on the basis of the EEK-2 emotional well-being questionnaire and ACR 2010 questionnaires The emotional well-being questionnaire is a patient-completed self-assessment scale for identifying major depression and anxiety disorders and their adjacent symptoms in the past month. The EEK-2 questionnaire consists of 28 statements formulated based on diagnostic criteria and clinical feasibility. The answers are on a scale of 0-4 and the subject indicates how much the problem has disturbed them in the last month. The ACR 2010 questionnaire is a questionnaire developed and validated by the American College of Rheumatology to assess the presence of the somatic symptom disorder and non-physical pain (such as fibromyalgia) in subjects. Fitness tests. Along with other data, the level of physical training of the conscripts is analysed using the standardised Army Physical Fitness Test (APFT), consisting of push-ups, sit-ups, and a 2-mile (3.2-km) run. The test has been adapted and used for a long time in the Estonian Defence Forces. Based on the results, the total score is calculated. Bone density study or densitometry. A mobile DXL machine is used to determine bone density. Bone density is determined from the heel bone. Bone density is determined twice during the study period on site. Bone density measurement of the heel bone is performed by densitometry with a DXL Calscan instrument (Demetech AB, SWE). The measurements are performed at the beginning of the study and after about 10 months, just before the end of the conscript. The procedure shows the bone density of the subjects. The second study provides an assessment of the effects of exercise on the musculoskeletal system. Health status monitoring. The health status of conscripts is monitored in the medical centre of the Combat Service Support Battalion of the 2nd Infantry Brigade, where records are made of the appeals and complaints of the conscripts, the number of days in inpatient care and bed rest, and the number of days when a conscript was absent from the conscript due to health problems. Evaluation of vitamin D content in food rations of the Defence Forces. The average daily vitamin D content of the 1-month period of conscription in the daily food ration for conscripts is assessed. Based on international analytical data on vitamin D content in food. Questionnaire completion and data collection, blood sampling, anthropometric evaluations, and health monitoring are performed by the study team.

Computed randomization will be used to divide conscripts into two groups; the intervention group, who received vitamin D3 capsules (4000 IU/100µg) or the intervention group, who received vitamin D3 capsules (600 IU/15µg) .

Prevalence of vitamin D measured serum vitamin D 25(OH)D concentration. Vitamin D considered as sufficient if the serum 25(OH)D concentration is ≥75 nmol/L, insufficient if <75 nmol/L, deficient if <50 nmol/L and critical deficiency < 25 nmol/L.

Prevalence of vitamin D measured serum vitamin D 25(OH)D concentration. Vitamin D considered as sufficient if the serum 25(OH)D concentration is ≥75 nmol/L, insufficient if <75 nmol/L, deficient if <50 nmol/L and critical deficiency < 25 nmol/L.

Prevalence of vitamin D measured serum vitamin D 25(OH)D concentration. Vitamin D considered as sufficient if the serum 25(OH)D concentration is ≥75 nmol/L, insufficient if <75 nmol/L, deficient if <50 nmol/L and critical deficiency < 25 nmol/L.

Prevalence of vitamin D measured serum vitamin D 25(OH)D concentration. Vitamin D considered as sufficient if the serum 25(OH)D concentration is ≥75 nmol/L, insufficient if <75 nmol/L, deficient if <50 nmol/L and critical deficiency < 25 nmol/L.

To assess subjective complaints related to the knee joint. The visual analogue scale VAS is used where '0' - no pain and '10' - maximum pain

To assess subjective complaints related to the knee joint. The visual analogue scale VAS is used where '0' - no pain and '10' - maximum pain

To assess subjective complaints related to the knee joint. The visual analogue scale VAS is used where '0' - no pain and '10' - maximum pain

To assess subjective complaints related to the knee joint. The visual analogue scale VAS is used where '0' - no pain and '10' - maximum pain

The subjects evaluate the subjective complaints and function of the knee joint with a questionnaire. It is possible to get a maximum of 100 points (no complaints) and a minimum of 0 points, which means extreme complaints.

The subjects evaluate the subjective complaints and function of the knee joint with a questionnaire. It is possible to get a maximum of 100 points (no complaints) and a minimum of 0 points, which means extreme complaints.

The subjects evaluate the subjective complaints and function of the knee joint with a questionnaire. It is possible to get a maximum of 100 points (no complaints) and a minimum of 0 points, which means extreme complaints.

The subjects evaluate the subjective complaints and function of the knee joint with a questionnaire. It is possible to get a maximum of 100 points (no complaints) and a minimum of 0 points, which means extreme complaints.

Is determined by isometric contraction while standing, upper limb away from the body, using a hand dynamometer (Lafayette Instrument Co., USA)

Is determined by isometric contraction while standing, upper limb away from the body, using a hand dynamometer (Lafayette Instrument Co., USA)

Is determined by isometric contraction while standing, upper limb away from the body, using a hand dynamometer (Lafayette Instrument Co., USA)

Is determined by isometric contraction while standing, upper limb away from the body, using a hand dynamometer (Lafayette Instrument Co., USA)

The Baecke Physical Activity Questionnaire consists of three parts and is based on a person's subjective assessment of their work, sport, and leisure. Each domain could receive a score from one to five points, thus allowing a total score from three (minimum) to fifteen (maximum).

International Physical Activity Questionnaire, which assesses the level of physical activity in the last 7 days. Categorical Score- three levels of physical activity are presented - low, medium and high.

International Physical Activity Questionnaire, which assesses the level of physical activity in the last 7 days. Categorical Score- three levels of physical activity are presented - low, medium and high.

International Physical Activity Questionnaire, which assesses the level of physical activity in the last 7 days. Categorical Score- three levels of physical activity are presented - low, medium and high.

International Physical Activity Questionnaire, which assesses the level of physical activity in the last 7 days. Categorical Score- three levels of physical activity are presented - low, medium and high.

Is done with the Cornell Musculoskeletal Discomfort Questionnaires where the intensity of pain and the region of the body can be noted as 'mild', 'moderate' and 'severe discomfort'.

Is done with the Cornell Musculoskeletal Discomfort Questionnaires where the intensity of pain and the region of the body can be noted as 'mild', 'moderate' and 'severe discomfort'

Is done with the Cornell Musculoskeletal Discomfort Questionnaires where the intensity of pain and the region of the body can be noted as 'mild', 'moderate' and 'severe discomfort'.

Is done with the Cornell Musculoskeletal Discomfort Questionnaires where the intensity of pain and the region of the body can be noted as 'mild', 'moderate' and 'severe discomfort'.

To assess pain in different regions of the body on a scale from 0 (none) to 10 (maximum possible pain)

To assess pain in different regions of the body on a scale from 0 (none) to 10 (maximum possible pain)

To assess pain in different regions of the body on a scale from 0 (none) to 10 (maximum possible pain)

To assess pain in different regions of the body on a scale from 0 (none) to 10 (maximum possible pain)

The assessment of subjective health status and health-related quality of life by the subject-RAND-36.

Is performed using standard questionnaires RAND 36. Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight. The lower the score the more disability.

The assessment of subjective health status and health-related quality of life by the subject- RAND-36.

Is performed using standard questionnaires RAND 36. Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight. The lower the score the more disability.

The assessment of subjective health status and health-related quality of life by the subject- RAND-36.

Is performed using standard questionnaires RAND 36. Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight. The lower the score the more disability.

The assessment of subjective health status and health-related quality of life by the subject- RAND 36.

Is performed using standard questionnaires RAND 36. Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight. The lower the score the more disability.

The assessment of subjective health status and health-related quality of life by the subject - Beck Depression

Is performed using standard questionnaires - Beck Depression Inventory. The BDI questionnaire is based on the assessment of the subject concerning aspects related to their mental health. Total score of 0-13 is considered minimal range, 14-19 is mild, 20-28 is moderate, and 29-63 is severe depression.

The assessment of subjective health status and health-related quality of life by the subject - Beck Depression

Is performed using standard questionnaires - Beck Depression Inventory. The BDI questionnaire is based on the assessment of the subject concerning aspects related to their mental health. Total score of 0-13 is considered minimal range, 14-19 is mild, 20-28 is moderate, and 29-63 is severe depression.

The assessment of subjective health status and health-related quality of life by the subject - Beck Depression

Is performed using standard questionnaires - Beck Depression Inventory. The BDI questionnaire is based on the assessment of the subject concerning aspects related to their mental health. Total score of 0-13 is considered minimal range, 14-19 is mild, 20-28 is moderate, and 29-63 is severe depression.

The assessment of subjective health status and health-related quality of life by the subject - Beck Depression

Is performed using standard questionnaires - Beck Depression Inventory. The BDI questionnaire is based on the assessment of the subject concerning aspects related to their mental health. Total score of 0-13 is considered minimal range, 14-19 is mild, 20-28 is moderate, and 29-63 is severe depression.

On the basis of the EEK-2 emotional well-being questionnaire. The emotional well-being questionnaire is a patient-completed self-assessment scale for identifying major depression and anxiety disorders and their adjacent symptoms in the past month. The EEK-2 questionnaire consists of 28 statements formulated based on diagnostic criteria and clinical feasibility. The answers are on a scale of 0-4 and the subject indicates how much the problem has disturbed them in the last month.

On the basis of the EEK-2 emotional well-being questionnaire.The emotional well-being questionnaire is a patient-completed self-assessment scale for identifying major depression and anxiety disorders and their adjacent symptoms in the past month. The EEK-2 questionnaire consists of 28 statements formulated based on diagnostic criteria and clinical feasibility. The answers are on a scale of 0-4 and the subject indicates how much the problem has disturbed them in the last month.

On the basis of the EEK-2 emotional well-being questionnaire.The emotional well-being questionnaire is a patient-completed self-assessment scale for identifying major depression and anxiety disorders and their adjacent symptoms in the past month. The EEK-2 questionnaire consists of 28 statements formulated based on diagnostic criteria and clinical feasibility. The answers are on a scale of 0-4 and the subject indicates how much the problem has disturbed them in the last month.

On the basis of the EEK-2 emotional well-being questionnaire.The emotional well-being questionnaire is a patient-completed self-assessment scale for identifying major depression and anxiety disorders and their adjacent symptoms in the past month. The EEK-2 questionnaire consists of 28 statements formulated based on diagnostic criteria and clinical feasibility. The answers are on a scale of 0-4 and the subject indicates how much the problem has disturbed them in the last month.

The ACR 2010 questionnaire is a questionnaire developed and validated by the American College of Rheumatology to assess the presence of the somatic symptom disorder and non-physical pain (such as fibromyalgia) in subjects. Pain score 0 no pain and 19 maximum pain. Symptoms score 0 - no symptoms and 12 maximum symptoms.

The ACR 2010 questionnaire is a questionnaire developed and validated by the American College of Rheumatology to assess the presence of the somatic symptom disorder and non-physical pain (such as fibromyalgia) in subjects. Pain score 0 no pain and 19 maximum pain. Symptoms score 0 - no symptoms and 12 maximum symptoms.

The ACR 2010 questionnaire is a questionnaire developed and validated by the American College of Rheumatology to assess the presence of the somatic symptom disorder and non-physical pain (such as fibromyalgia) in subjects. Pain score 0 no pain and 19 maximum pain. Symptoms score 0 - no symptoms and 12 maximum symptoms.

The ACR 2010 questionnaire is a questionnaire developed and validated by the American College of Rheumatology to assess the presence of the somatic symptom disorder and non-physical pain (such as fibromyalgia) in subjects. Pain score 0 no pain and 19 maximum pain. Symptoms score 0 - no symptoms and 12 maximum symptoms.

the level of physical training of the conscripts is analysed using the standardised Army Physical Fitness Test (APFT). Soldiers were scored based on their performance in three events consisting of the push-up, sit-up, and a two-mile run, ranging from 0 to 100 points in each event. A minimum score of 60 in each event was required to pass the test. The soldier's overall score was the sum of the points from the three events. If a soldier passed all three events, the total could have range from 180 to 300.

the level of physical training of the conscripts is analysed using the standardised Army Physical Fitness Test (APFT). Soldiers were scored based on their performance in three events consisting of the push-up, sit-up, and a two-mile run, ranging from 0 to 100 points in each event. A minimum score of 60 in each event was required to pass the test. The soldier's overall score was the sum of the points from the three events. If a soldier passed all three events, the total could have range from 180 to 300.

the level of physical training of the conscripts is analysed using the standardised Army Physical Fitness Test (APFT). Soldiers were scored based on their performance in three events consisting of the push-up, sit-up, and a two-mile run, ranging from 0 to 100 points in each event. A minimum score of 60 in each event was required to pass the test. The soldier's overall score was the sum of the points from the three events. If a soldier passed all three events, the total could have range from 180 to 300.

The health status of conscripts is monitored in the medical centre of the Combat Service Support Battalion of the 2nd Infantry Brigade, where records are made of the appeals and complaints of the conscripts, the number of days in inpatient care and bed rest, and the number of days when a conscript was absent from the conscript due to upper respiratory infection (UTRI).

The health status of conscripts is monitored in the medical centre of the Combat Service Support Battalion of the 2nd Infantry Brigade, where records are made of the appeals and complaints of the conscripts, the number of days in inpatient care and bed rest, and the number of days when a conscript was absent from the conscript due to musculoskeletal injury.

Inclusion Criteria: Participating in military service at the Estonian Defence Forces Voluntary participation in the study Exclusion Criteria: Refusal of a conscript or withdrawal of consent Departure from military unit Force majeure Acute trauma or illness in the course of conscript requiring hospital treatment Serious side effects or allergies to vitamin D capsules

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