The study sample consisted of 74 combat male recruits from an elite combat unit in the IDF. Volunteers were recruited at the beginning of a mandatory three year military service program. All volunteers had successfully completed a strenuous 4-day sorting course 3 months prior to their induction. The military training protocol was designed to prepare the soldiers for combat missions, and included general physical fitness, physical work under pressure, hand to hand combat training, direct fire battle, leadership development and stressful combat conditions.
The study was approved by both Institutional Review Board Committees of the IDF and Sheba Medical Center, and the U.S. Army Research Institute of Environmental Medicine at Natick, MA, and all the participants signed informed consent for participation in the study.
Data on the soldiers' anthropometric measurements, nutritional habits, iron indices, and serum calcium were collected on induction and again after 4 months. Blood samples were also taken 6 months after induction. A medical evaluation was conducted at baseline and then bi-weekly during the 6-month period by two orthopedic surgeons in order to detect the presence of stress fracture and other overuse injuries.
Anthropometric measurements included body weight, height, body fat percentage and calculation of body mass index (BMI). Height (cm) was measured using a stadiometer (± 1 cm) and body weight (kg) was determined with a metric scale (± 100 gr). In order to avoid errors, the same researcher completed all anthropometric measurements at each data collection point. Body fat percentage was calculated according to Siri from a 4-site skin fold thickness (biceps, triceps, subscapula, and suprailiac)  using Lange skin fold calipers (Beta Technology, Santa Cruz, CA).
Food intake was assessed using the food frequency questionnaire (FFQ), developed and validated for the Israeli population by The S. Daniel Abraham International Center for Health and Nutrition at the Ben-Gurion University, Israel [23, 24]. It is a long-term dietary assessment tool consisting of 126 food items divided into nine food groups that can be analyzed for nutrient and food group intake, such as: 1) eggs, milk, and milk products; 2) fats (including sauces); 3) chicken, meat, and fish; 4) bread and baked products; 5) starches and legumes; 6) fruit; 7) vegetables; 8) snacks and cookies; and 9) beverages. Subjects completed the FFQ with the assistance of a dietitian at two time points: on induction, referring specifically to the previous 6 months, and then again 4 months after starting BT, referring to the 4 months of BT. All food input was to be reported . The FFQ analysis produces daily nutritional intake of proteins, carbohydrates, total fat, iron, folate, vitamins (D, B6, and B12), calcium, zinc, and magnesium. Our study referred to the MDRI and NSOR and we compared both raw data and data normalized for body weight.
Hematology and blood chemistry
Blood samples representing nutritional status were collected at three time points (0, 4, 6 months) and the following components were analyzed: hemoglobin (Hgb), iron, transferrin, ferritin, folic acid, vitamin B12, and calcium. Approximately 30 cc's of venous blood samples were obtained by antecubital venipuncture into tubes (BD Vacutainer; Becton, Dickinson and Company®, 2002 BD) containing the appropriate anticoagulant. All samples were taken during the morning hours (0600-0700 h), in a sitting position after an overnight fast (6-10 h) and no exercise. The samples were placed in ice and sent within four hours to be processed and analyzed at the Sheba Medical Center Laboratories (Hematology and Biochemistry). Blood counts were performed on fresh blood using an automated analyzer (Cell-Dyn® 3000; Abbott Diagnostics, Abbott Park, IL) for measuring Hgb values. Serum ferritin was measured using an electrochemiluminescence immunoassay (Roche Elecsys®, Roche Diagnostics GmbH, Mannheim, Germany, reference: of 16-293 ng/ml). Serum iron was measured with a commercial kit on Olympus (AU2700, reference ranges 60-170 μg/dL). Vitamin B12 and folic acid levels were determined with an automated analyzer (Architect Abbott Diagnostics). Serum transferrin was measured with an immunoturbidimetric assay (Tina-quant® with Roche Diagnostics GmbH, Mannheim, Germany, reference ranges 193-378 mg/dL). Transferrin saturation was calculated according to the following formula: transferrin saturation (%) = serum iron/serum transferrin. Blood calcium was measured using a commercial kit on Olympus (AU2700, reference values: 8.5-10.9 mg/dl). Radioimmunoassay (RIA) was used to measure 25(OH)D levels (DiaSorin, Stillwater, MN, reference range 30.0-74.0 ng/ml). Parathyroid hormone (PTH) was measured by immunoassay with chemiluminescent detection on the Immulite 2000 (Diagnostics Products Corporation, Los Angeles, CA, reference range 12.0-72.0 ng/L).
Hematological deficiencies were established as follows: anemia was diagnosed at Hgb levels of less than 14 g/dl, and ferritin levels (< 20 ng/ml).
Nutrition provided to recruits
The recruits had 3 main meals and 3 snacks a day. Breakfast (7-8 am, about 2 h after awakening), which included porridge, bread, 1-2 eggs, cream cheese, vegetables, olives, jam and additional savory spread for the bread (avocado, chickpea etc, depending on the season). A chocolate drink (200 ml milk) and milk desserts were also served. Dinner (12-1 pm) included soup, a meat/chicken/fish portion (200 grams) 2 salads and a cooked vegetable, a starch (potatoes/rice/macaroni), bread and a fruit desert. Supper (5-6 pm) included a main portion (1-2 eggs/macaroni etc) bread, cream cheese and spreads, hard cheese, and all other components served at breakfast. The three intermediate snacks were usually 1-2 sandwiches with jam or chocolate spread. All food was provided at no cost to the recruits.
Dietary supplements were not given or encouraged, though they were not prohibited and their use was not monitored. Formally, recruits were allowed to get additional snacks at the canteen, but they were not given access to the canteen on a regular basis. They might also have eaten extra food sent by relatives.
Injury surveillance and bone stress injury diagnosis took place over the course of the entire 6-month training period. We used three sources of data: the unit physicians treating the recruits recorded overuse injuries separately in a personal surveillance table. Two orthopedic surgeons examined the recruits every 2-3 weeks and registered their findings in the recruits' central army Computerized Patient Record (CPR). Stress reactions and fractures were diagnosed by clinical examination and confirmed by radiography or bone scintigraphy . Sixty two recruits without clinical signs of stress reactions and those whose imaging ruled out a stress reaction or fracture were classified as the NSF group. Twelve recruits with stress fractures of the tibia or femur confirmed by imaging were classified as the SF group. Since the mechanism for developing stress fractures in the metatarsals is fatigue and not remodeling, as in the long bones , we focused only on stress fractures of long bones.
Data analysis was performed using the Statistical Package for the Social Sciences software version 15.1 (SPSS INC., Chicago, IL). Comparisons between study groups over the time points, and at each phase were performed using repeated measures ANOVA (groups and time; α < 0.05) followed by pairwise comparisons using Student's t-test with adjustments for multiple comparisons by Tukey-Kramer. Analysis of the nutritional data produced descriptive statistics including mean, standard deviation, standard error, and range.