The goal of this clinical trial is to learn whether oral iron supplementation will increase skeletal muscle iron storage and its effects on exercise capacity in females with suboptimal iron status. The study will include healthy females, ages 18-40, who either have suboptimal or optimal iron stores.

The main questions it aims to answer are:

• Does iron supplementation increase skeletal muscle iron storage and alter the expression of iron-related and mitochondrial proteins?
• Does iron supplementation improve single-leg exercise performance?
• Is serum ferritin correlated with the abundance of iron-related proteins in skeletal muscle?

Researchers will compare outcomes from females with suboptimal iron status who receive oral iron supplementation to those who receive a placebo to see if supplementation improves muscle iron storage, protein expression, and exercise performance. Additionally, a non-intervention control group with optimal iron status will be included to assess baseline differences.

Participants will:

- Be randomly assigned to receive 150 mg elemental iron or placebo (maltodextrin) every other day for 12 weeks

Complete pre- and post-supplementation testing, including:

• Blood draws to assess iron status
• Skeletal muscle biopsies to analyze protein content
• Whole-body and single-leg exercise tests to assess performance
• Controls will undergo baseline-only testing to compare physiological and biochemical markers

This study is intended to evaluate the effects of oral iron supplementation on muscle iron levels in females with suboptimal iron status. The design involves both a randomized controlled trial (RCT) for those with low blood iron levels, as well as a cross-sectional component, including only healthy controls. Iron supplementation will be administered to half of those enrolled in the RCT portion to evaluate the effects of iron uptake within the muscle. Therefore, the primary goal of the study is to determine if iron is stored within the muscle in meaningful amounts following an extended period of oral iron supplementation in healthy females with suboptimal iron status.

The study's rationale extends from the high prevalence of low iron levels among females globally, especially among those who are involved in endurance sports. At the level of the muscle, iron serves in several key biological functions, including oxygen transport, energy production, and even recovery from exercise, making it essential for performance. Although the investigators understand some amount of iron is stored within the muscle, the investigators are currently unaware whether meaningful changes occur following a period of supplementation. Therefore, the proposed protocol outlined below is designed to test the hypothesis that taking iron for a prolonged period at adequate doses will result in meaningful changes.

Participants involved in this study will include biological females aged 18-40 with suboptimal iron status (blood ferritin ≤50 µg/L), who are otherwise healthy, for the RCT portion, or normal iron status (blood ferritin ≥50 µg/L) for healthy controls. Interested participants will be screened, and those eligible for the RCT will be randomly assigned to either an oral iron supplement or a placebo sugar pill, ensuring that the encapsulation is preserved to promote blinding. Participants will consume the assigned treatment once every 2nd day for 12 weeks. Exercise and other biological tests will be performed both before and after supplementation during the RCT, while healthy controls will only participate in baseline testing.

The following is an overview of the study design:

Baseline testing

This phase involves five visits and approximately 6.5 hours of laboratory testing spread over two weeks. Participants in the RCT and those acting as healthy controls will both complete the baseline testing phase.

Visit I: Blood draw

Participants will report to the laboratory in the fasted condition. They will be verbally informed of the methods of this study by the researcher and given time to ask questions before their written consent is obtained. Participants will complete a short survey to characterize their menstrual cycle. Subsequently, an individual trained in phlebotomy will collect venous blood for analysis from an antecubital vein using appropriate techniques. Following analysis of the blood iron markers, participants will be informed whether they are eligible to participate in the RCT or serve as a control. This visit will take approximately 1 hour.

Visit II: Body composition scan and familiarization

Participants will arrive fasted and undergo a body composition scan via dual-energy x-ray absorptiometry (DXA), which takes approximately 10 minutes. They will then be permitted to eat and drink. Participants will complete additional questionnaires (to be repeated before each exercise testing session) detailing perceived rest and food records. They will then be familiarized with a dynamic knee extension (i.e., kicking) task involving 15 minutes of exercise at varying intensities with feedback on technique. Once familiarized, participants will perform a single-leg incremental ramp protocol to determine their quadriceps peak power. In this test, participants will kick at a fixed cadence (e.g., 40 kicks per minute) while resistance gradually increases until they are unable to continue due to fatigue or personal choice. This test will last approximately 10 to 20 minutes. This visit will take approximately 1.5 hours.

Visit III: V̇O₂max and familiarization

On a separate day, at least 24 hours after the last visit, participants will return to the lab without fasting. They will complete an incremental ramp exercise test on a cycle ergometer (double-leg) to measure V̇O₂max, characterizing their aerobic fitness. This test involves gradually increasing resistance until the participant can no longer continue. After a rest period of at least 20 minutes (or longer if needed), participants will perform additional familiarization with the dynamic knee extension exercise, consisting of 30 minutes of kicking at various intensities. This visit will take approximately 1.5 hours.

Visit IV: Lower-body power, quadriceps oxidative capacity, and critical power

On a separate day, at least 24 hours after their last visit, participants will return to the lab without fasting. During this visit, participants will perform countermovement jumps and undergo testing of quadriceps oxidative capacity (OxCap) and critical power (CP) in their dominant leg. They will begin with OxCap testing, which involves a brief isometric contraction of the knee extensor muscles followed by repeated short arterial occlusions using a rapid cuff inflator. This test is performed in duplicate. Next, participants will perform countermovement jumps - a series of vertical jumps on a force plate repeated three times with 60-second rests in between. After resting for 10 to 15 minutes, they will perform three bouts of dynamic knee extension at different intensities to task failure to determine CP. Each bout will last 2 to 20 minutes and be separated by at least 20 minutes of rest. This visit will take approximately 2 hours.

Visit V: Muscle biopsy procedure

On a separate day, at least 24 hours after their last visit, participants will return to the lab in a fasted state for a muscle biopsy. A physician will obtain a small muscle sample using the proper technique, which will later be analyzed for iron-related and mitochondrial protein content. This visit will take approximately 45 minutes.

Supplementation phase

This phase involves two visits and approximately one hour of laboratory testing. Only participants in the RCT will complete this phase.

Participants in the RCT will be randomized to receive either an iron supplement or a placebo in a 1:1 ratio using a concealed allotment procedure. Participants will consume either one iron pill (150 mg elemental iron) or one placebo pill (PolyCal, maltodextrin) every other day for 12 weeks. Each iron supplement contains 50 mg of elemental iron, while the placebo includes none. This dosing protocol is based on prior research showing that alternate-day dosing improves gastrointestinal tolerance. Both iron and placebo pills will be encapsulated within a gelatin capsule for blinding, ensuring no difference in release rate.

Visits VI and VII: Week 4 and 8 blood draws

Participants will visit the lab during weeks 4 and 8 of their supplementation period to provide blood samples. Before each blood draw, they will complete questionnaires about gastrointestinal symptoms and a food record covering the previous three days. Blood will be collected by a trained individual using a standard venipuncture technique. Each visit will take approximately 30 minutes.

Post-supplementation testing

Post-supplementation testing will involve the same procedures as baseline testing, but the visits will be slightly more condensed and re-ordered. In total, participants will visit the lab three times over approximately two weeks for about 5.5 hours of testing. Only RCT participants will complete this phase.

Visit VIII: Blood draw, muscle biopsy, and body composition scan.

After completing supplementation, participants will return to the lab in a fasted state to provide a blood sample and a muscle biopsy, and to undergo a body composition scan. Participants will also repeat familiarization with the single-leg exercise task, using the same procedure from visit II. They will complete questionnaires on gastrointestinal symptoms and their three-day food record. This visit will take approximately 2 hours.

Visits IX and X: Exercise testing

These visits are identical to visits III and IV from the baseline phase and will take the same amount of time (1.5 and 2 hours, respectively) over approximately one week. They will begin a minimum of 48 hours after visit VIII and be separated by at least 24 hours. Before each test, participants will complete questionnaires about rest and their food records for the preceding three days. After all testing is complete, participants and researchers will record their guess about which supplement was consumed to assess the effectiveness of blinding. Participants will then be informed of their group allocation.

Inclusion Criteria:

• biologically female
• between the ages of 18-40 years of age at the time of enrolment
• plasma ferritin ≤ 50 µg/ L (RCT) or >50 µg/L (controls)
• complete Get Active Questionnaire (GAQ: Canadian Society for Exercise Physiology) without contraindication

Exclusion Criteria:

• have a body mass index (BMI) > 30 kg/m2
• taking medications that have been shown to affect cardiovascular, respiratory, or metabolic responses to exercise (e.g., β-blockers, anti-inflammatories, anti-coagulants, insulin, etc.)
• hemoglobin ≤ 120 g/L (anemia cut-off for women)
• have known health problems or contraindications that prohibit exercise, including renal or gastrointestinal disorders, metabolic disease, heart disease, vascular disease, arthritis, diabetes, respiratory disease, uncontrolled blood pressure, dizziness, thyroid problems, or any other health conditions that are being treated and deemed likely to confound the results
• dieting for weight loss or following a low-carbohydrate diet (known to affect iron homeostasis)
• history of smoking or using tobacco products
• consuming excessive amounts of alcohol (>21 units/week)
• supplementing with iron (>5mg/day) in the last 3 months
• any blood donation or surgeries in the past 3 months
• antibiotic use within the previous four weeks before study enrolment
• current laxative use
• known history of thalassemia
• known bleeding disorder
• chronic use of nonsteroidal anti-inflammatory drugs
• Currently/last 3 months taking prescription medications that are known to affect iron absorption (i.e. Antacids/PPIs (e.g., omeprazole), H2 Blockers (e.g., ranitidine), Tetracycline Antibiotics (e.g., doxycycline), Quinolone Antibiotics (e.g., ciprofloxacin), Cholestyramine, Colchicine, Methyldopa).
• currently pregnant or plans to get pregnant within the duration of the study, and
• do not understand English
• Participants will be removed from the study if they begin taking any other forms of supplemental iron or choose to donate blood