Background: Relative Energy Deficiency in Sport (REDs) describes impairment of health and performance due to problematic (long-term/severe) low energy availability (LEA), with or without eating disorders. LEA is frequently reported in sports with high training volumes, especially in leanness demanding sports, and 20% of female and 9% of male Norwegian national team athletes have been reported to have eating disorders. Potential trigger factors are e.g., dieting, injuries, coaching behavior, and subculture aspects e.g., focus on low body weight. The main questions that will be addressed are: 1. What is the prevalence of eating disorders and REDs among Swedish elite athletes and controls? 2. What is the impact of problematic LEA on health and performance aspects in both male and female athletes? Methods: National team athletes and gender and matched controls will be invited to an anonymous on-line survey. Elite athletes who agree to participate, will be invited to assessment of eating disorders, nutritional and physiological status (e.g., metabolic and endocrine markers, bone health, microbiota, dietary intake, energy availability, and performance).

The syndrome of REDs describes impairment of physiological functions e.g., reproduction and bone health with negative impact on performance. The etiology behind is problematic low energy availability (LEA), defined as low energy intake relative to exercise energy expenditure relative to fat free mass. Eating disorders can either proceed or result from REDs. LEA induces metabolic and endocrine alterations, and energy balance at a lower level prevents further weight-loss. Therefore, most athletes with LEA have a body weight within the normal range, and low resting metabolic rate is a recognized LEA surrogate marker.

LEA suppresses anabolic hormones e.g. estradiol/testosterone, while cortisol levels increase, and bone formation markers are reduced. Low testosterone levels have been reported in male endurance athletes, with potential negative effects on fertility. Among Irish male athletes from different sports 23% were identified with low libido and associated with increased injury risk. Menstrual disturbances due to LEA is frequently reported, and is associated with low bone mineral density and injuries.

Glucose is an important substrate for the muscles and an essential substrate for brain neurons and immune cells. Low glucose availability is considered a major trigger to the alterations of the hypothalamic-pituitary axis hormones causing suppression of the sex and thyroid hormones and increase in cortisol. Blood glucose homeostasis is therefore protected with several mechanism (hepatic gluconeogenesis, increased lipolysis and ketogenesis) during prolonged LEA. Hence, hypoglycemia is a marker of a prolonged and severe catabolic state.

LEA has been associated with gastrointestinal problems in female athletes, derived from hormonal and dietary factors affecting motility and digestion. Long-term LEA in patients with anorexia nervosa is associated with profound alterations of the gut microbiome, with negative impact on gastrointestinal function and immunologic processes, and a reduced microbial diversity is therefore suggested as a potential useful biomarker of REDs in athletes. It is therefore relevant to investigate the associations between the gut microbiome, Gastrointestinal function and immunology status in athletes with and without clinical indication of LEA.

LEA affects both men and women, athletes as well as recreational active people however, the threshold and endocrine response seem to differ between gender. Although menstrual disturbances has been frequently reported in female athletes for decades, few studies have investigated the impact on performance, and no study has yet investigated the performance effects of low libido in male athletes. Furthermore, no study has investigated microbial diversity in male and female athletes with clinical symptoms of LEA. Therefore, it is highly relevant to investigate aspects of physiology, nutrition, performance, and sport environment regarding REDs and eating disorders in both male and females.

Objective: The aims of the study are to investigate the prevalence of athletes at risk of eating disorders and REDs among Swedish athletes and controls, and to investigate physiological aspects of elite athletes with and without problematic LEA. Furthermore, to investigate if there is a difference in nutritional status (metabolic and endocrine markers, iron- vitamin D status, energy availability, glucose homeostasis, resting metabolic rate, body composition including bone mineral density), gut microbiota (including associations to immunity), immune function, and performance, in athletes diagnosed with REDs according the International Olympic Committee REDs Clinical Assessment Tool vs. gender matched athletes without REDs. An additional aim is to evaluate if problematic LEA affects biological markers in the biological passport also called "Athlete Biological Passport" in anti-doping.

Methodology and plan of work National senior team and recruiting squad athletes from Swedish sport federations will be invited to an anonymous on-line survey. The 20 minute on-line survey includes: Eating Disorder Examination Questionnaire (EDE-Q). An EDE-Q global score ≥ 2.3 for females and ≥ 1.68 for males indicate risk of EDs; Exercise Addiction Inventory (EAI). A total EAI-score 24-30 indicate compulsive exercise; LEA in Females Questionnaire (LEAF-Q) and LEA in Males Questionnaire (LEAM-Q) to categorize athletes ar risk of LEA; Major Depression Inventory (MDI) to detect depression symptoms; Swedish Acceptance and Action Questionnaire (SAAQ) to assess psychological flexibility; and Motivation to Change Questionnaire (MCQ) to asses the ability for behavioral changes.

Participants who are interested in participating in an on-line diagnostic eating disorder examination interview (the EDE-16) and/or physiological testing, provide their name, cell-phone number, and e-mail address after completing the online survey, and will thereafter be invited to the sport physiology lab for the next phase of the study (physiological assessments).

On the day of the physiological examination, the participants will be asked to deliver a fecal sample. Gut microbiota composition bacterial DNA will be sequenced using next-generation sequencing of microbial genes.

Fasting resting metabolic rate will be measured, using a ventilated open hood system. The ration between measured and predicted resting metabolic will be calculated, and low resting metabolic rate will be defined as resting metabolic rate ratio < 0.90.

Blood samples will be collected and stored (-80°) for later analyses according to standard laboratory procedures (e.g. iron- and vitamin D status, hormones, metabolic, bone and immune markers).

Body weight, height (fixed stadiometer), fat free mass, fat mass and bone mineral density (whole body, femur, and lumbar spine) will be measured using dual-energy x-ray absorptiometry. Low bone mineral density will be defined as Z score -1 to -2 in at least one measured site and osteoporosis as Z-score ≤-2.

After a standardized meal and warm up, a bicycle ergometer exercise test will be performed (6 minutes at 50 watt, followed by 12-14 watt per minute increased workload until exhaustion). Heart rate and an air-tight mask covering the mouth and nose will be used to measure peak oxygen uptake. Capillary lactate will be drawn before, direct after and 5 minutes after the test. Explosive lower body power will be assessed through a counter movement jump-test. Isometric mid-thigh pull will be performed to assess whole body strength and force production. A Wingate-test will be performed to assess muscular power and anaerobic capacity. Post exercise blood-samples will be drawn and stored (-80°) for later analyses according to standard laboratory procedures (e.g., immune markers).

Participants will be asked to wear a subcutaneous continuous blood glucose monitor, an accelerometer, throughout the day except during training where they will wear heart rate monitors and register all training sessions in their home environment for 7 consecutive days. During 4 days in the the same period they will be asked to register all food intake using an electronic kitchen scale and an on-line nutrient program while information of energy and nutrient intake is blinded for the participants. Energy and nutrient intake will be calculated using a nutrient analysis program. Training heart rate and training logs will be used to assess exercise energy expenditure based on individual prediction equations from measured heart rate and corresponding energy expenditure during the peak oxygen uptake test. Energy availability will be calculated by subtracting mean exercise energy expenditure from mean energy intake relative to fat free mass.

Eumenorrheic athletes will be tested on day 2-6 of menstruation, while other athletes will be tested when convenient. All materials (electronic kitchen scale, heart rate monitor, fecal sample collective tube and a prepaid security envelops for returning materials) will be provided.

The skills and equipment for all assessments are available at the Linnaeus University including preparation, storage, and analysis of blood. Fecal samples will be analyzed at the Department of Nutrition, Exercise and Sport, University of Copenhagen.

Power calculation: to provide 80% power to detect a clinically relevant difference in sex hormones a minimum of 14 females (estradiol standard deviation 79.0 nmol/L) and 8 males (total testosterone standard deviation 7.4 pmol/L) in each group will be included.

Recruitment will be performed via the Swedish Olympic Committee, the national sport federations, and social media. The on-line surveys will be launched via the REDs Sweden project webpage and will be open March 2023 to June 2024. Recruitment of age and gender matched controls to the online survey will be performed via social media. Athletes who agree to participate in physiological testing will be continuously scheduled for tests and assessments.

Ethical reviews: The study will follow the checklist issued by the national committees for research ethics and the Helsinki Declaration. Ethical approval has been approved (2021-07031-01). All information and results will be coded (anonymized) before analysis and stored according the general data protection regulation. A list with codes connecting participant names and information will be kept safely locked in by the principal investigator at the Linnaeus University. There are no expected risks with participation. The radiation during the Dual-energy X-ray absorptiometry measurement is negligible. Participants may feel uncomfortable during the assessment of resting metabolic rate and blood sampling. The study collaborators will make sure that participants feel safe during all measurements. If a diagnosis is identified, participants will be referred to the health care system. Participants will be informed regarding ethical rights, the project, expected time cost for participation, and feedback on individual results.

Inclusion:

• National-team level athletes
• Non-elite athletic controls

Exclusion (physical assessment):

• chronic illness (e.g., diabetes, hypothyroidism)
• injury
• smoking
• pregnancy
• hormonal contraceptive use

Swedish male and female national team athletes and gender- and age matched controls.