The goal of this clinical trial is to understand how the body responds to short-term, severe low energy availability (LEA) in healthy, weight-bearing endurance athletes aged 18-45 years old. LEA describes a mismatch between an individual's dietary energy intake and the energy cost of their commitments for training and competition. The main questions this trial aims to answer are:

1. What effect does short-term, severe LEA have on sleeping metabolic rate?
2. What effect does short-term, severe LEA have on other body systems identified within the Relative Energy Deficiency in Sport (REDs) Health and Performance Conceptual models?

Researchers will compare a control trial with both a LEA trial achieved through diet restriction and a LEA trial achieved through increased exercise to see if there are differences in the body's response.

Participants will complete three 6-day trials, a minimum of 3-weeks apart, involving:

• Prescribed diet (all food provided)
• Prescribed running and/or cycling exercise
• Two visits to ACU Fitzroy campus for blood tests and exercise testing
• 50 hour stay (two nights and two days) in the ACU metabolic chamber

The study will involve a three-sequence crossover design, with participants undertaking a control trial first, involving energy availability of 40 kcal/kg fat free mass (FFM) and a 15 kcal/kg FFM exercise energy expenditure (EEE) as baseline training volume, before a counterbalanced allocation to low energy availability via dietary restriction (LEADIET) or low energy availability via increased exercise energy expenditure (LEAEX). This design will allow further screening of Relative Energy Deficiency in Sport (REDs) risk, identify participants who are likely to be unable to fulfil the exercise commitments of LEAEX and avoid the unknown carryover effects of two consecutive low energy availability (LEA) trials on the subsequent control trial, as would occur in some participants with a fully counterbalanced treatment allocation.

Baseline testing (body composition/bone mineral density via DXA, aerobic capacity, LEA hormone blood panel, vitamin D, resting metabolic rate, REDs screening via REDs-CAT2) will be undertaken to confirm inclusion and develop individualised diet and training plans. Thereafter, participants will undertake a 6-day supervised exposure to energy availability of ~40 kcal/kg FFM/d, involving supplied meals and prescribed daily running/race walking. For female participants, trials will commence within days 1-5 of the menstrual cycle, to ensure that all testing occurs in the same (follicular) phase. A minimum 3-week washout period will occur between trials, with participants being guided to return to habitual training and dietary habits. Trials 2 and 3 will consist of exposure to severe LEA (15 kcal/kg FFM/d), which is achieved either by superimposing restricted energy intake (EI) or increased (doubled) EEE on the previous training plan.

Diet plans and food provision will be managed by experienced sports dietitians to incorporate individual preferences and intolerances. While participants are free-living at the start of each trial week, food drops will be undertaken to check well-being and compliance to the diet/ training intervention. The final 2 days of residence at ACU facilities (medical unit and metabolic chamber) will allow more comprehensive data collection. Individual protocols (described briefly below) have been previously developed by our group and collaborators. Together, they will investigate within and between trial changes in metrics representing the range of body systems that have been identified at risk of perturbations due to LEA within the REDs Conceptual Health and Performance Models.

Summary of the protocols completed in this study:

1. VO2 max: Running protocol: Measured once at screening visit (and potentially remeasured if participant trials are spaced >3 months apart and aerobic fitness requires rechecking)

2. Resting Metabolic Rate: Outpatient protocol via Parvo metabolic cart: Measured once in baseline testing to enable calculation of EA components for intervention

3. DXA: total body composition: Measured once at screening visit to enable calculation of EA components for intervention, and twice in each trial (Day 1 and Day 5)

4. DXA: bone mineral density (Hip and Spine): Measured once at screening visit

5. Cognitive tests: 5-minute psychomotor vigilance test (PVT) & 3-minute Simon Squared, delivered via Inquisit software (screening visit, then Day 1 and Day 6)

6. RESTQ-76: questionnaire of overall wellbeing (Day 6)

7. Diet feedback questionnaire (Day 6)

8. Oura Ring: sleep characteristics, resting heart rate (HR), total energy expenditure (EE), basal body temperature, HR variability (HRV) (throughout each trial)

9. Somfit: sleep architecture (Night of Days 3, 4, and 5)

10. Training capacity: Garmin-derived HR and EE, rate of perceived exertion (RPE, throughout trial), metabolic, physiological and perceptual responses during standardised running test (Day 5)

11. Dietary iron absorption and incorporation: Intake of stable isotope (3.8mg, Fe57 as ferrous sulphate) (evening of Day 4), with 8 blood samples collected from 0-480 min post dose to assess iron absorption. A further blood sample taken after 28 days will measure incorporation into red blood cells to assess functional iron availability

12. Venepuncture/Venous cannulation: Total blood volume per trial = 140 ml for:

    • LEA panel: testosterone, estrogen, progesterone, IGF-1, T3, leptin, iron status, lipids, glucose/insulin (screening visit, Day 1 and Day 5)
    • Hormone pulsatility: 8 h @15 min collection of Luteinising Hormone, Growth Hormone, Cortisol (overnight Day 4)
    • Appetite peptides: Leptin, Active Ghrelin, PP, GLP-1, PYY (Day 5)
    • Immune response: IL-6, mitochondrial respiration, reactive oxygen species emission, and protein expression in peripheral blood mononuclear cells (PBMCs), circulating leukocyte type distribution and mobilisation, circulating cell-free DNA (Day 5)
    • Bone turnover: CTX-1 and P1NP (Day 5)
    • Iron absorption and response: plasma iron-isotope appearance (overnight Day 4), Hepcidin (Day 5)

13. Exercise metabolism: blood lactate, glucose (finger prick collection, Day 5 exercise)

14. Gut comfort: Standardised GI questionnaire pre- and post-exercise (Day 5 exercise)

15. Protein turnover: Consumption of stable isotopes of 15N-glycine and D3-3-methyl-histidine (Day 4), with a baseline urine sample, and serial timed samples (Days 5 and 6) will allow determination of whole-body protein turnover (measured as 15N-Alanine), and muscle proteolysis

16. Bone breakdown: Measurement of Ca isotope abundance in urine (on waking urine sample on Day 1 and 24-hour urine sample on Day 5/6 in metabolic chamber) will allow assessment of bone breakdown (periods of greater bone breakdown: formation will see greater abundance of lighter isotopes).

17. Metabolic chamber: 24 h energy expenditure, sleeping metabolic rate, basal metabolic rate, EEE (Day 5/6 in metabolic chamber)

18. Blood glucose monitoring: blood glucose monitoring, using continuous glucose monitors (Freestyle Libre Pro iQ) (from Day 1 through to end of Day 6)

Inclusion Criteria:

• 18-45 years old
• Well trained weight-bearing athlete (tier 2-4 1)
• Perform ~45km per week of weight-bearing endurance training, and able to undertake the prescribed exercise in each trial condition
• Pass the ESSA pre-exercise screening tool and/or obtain GP clearance to exercise

Exclusion Criteria:

• Assessment of red status on the REDs Clinical Assessment Tool
• Unable to attend ACU in Fitzroy, Victoria, for the 6 study visits for completion of the study protocol
• Pregnancy
• Use of hormonal contraceptive with the previous 3 months
• Onset of peri/menopause

The data that support the findings of this study will be available from the corresponding author for a period of 36 months from publication, upon reasonable request for academic use. The data will not be made publicly available, as it contains information that could compromise research participant consent.

For a period of 36 months from publication of the primary outcome.