Design and detailed description of methodology This is a parallel-group randomized controlled trial, blinded to the 17β-estradiol (n=10) or placebo (n=10) supplement administered every day for 10 days. Twelve healthy young men (age: 18-25yrs; BMI: 22-33 kg/m2) will be recruited to participate in this study. During phone or e-mail conversations we will explain the purpose and experimental design of the study and address all questions the participants might have (see telephone and email scripts). Participants will visit the laboratory for a screening to assess whether they are eligible to participate in this study. During the screening we will first explain the experimental design and obtain written informed consent. A medical questionnaire will be completed by the participants to assess their general health and use of medication. Body height and mass will be measured as baseline characteristics of the participants. Participants will be requested to fill out a 3-d dietary record to assess habitual energy and protein intake. Participants will be randomly assigned to the 17β-estradiol (n=10) or placebo (n=10) supplementation group. Randomization will be performed using a computerized list randomizer (https://www.random.org/lists/), and participants will be sequentially allocated to a condition according to the randomized list. The experimental trial is composed of 10 days of 17β-estradiol (Estrace, Acerus Pharmaceuticals Corporation, Mississauga, ON, Canada or placebo (400 mg/day Polycose, Abbott Laboratories, St. Laurent, QC, Canada) supplementation, including 3 days of pre-immobilization, and 7 days of single leg immobilization. Three days prior to immobilization (t=-3 d) a blood sample will be obtained and muscle strength will be assessed before daily 17β-estradiol or placebo supplementation will be initiated. Participants will consume 1mg/day for 3-days and 3mg/day for 7-days of Estrace or 400 mg/day of Polycose for 7-days. The estrogen and placebo bottles are coded to ensure complete blinding to both researchers and participants. Additional blood samples will be obtained to assess the compliance of the participants to the supplementation. In addition, at t=-3 d a saliva sample will be obtained before ingesting 150 mL of deuterium-labeled water (D2O). Daily saliva sampling will be continued until the end of the experimental trial to assess precursor pool enrichments. Dietary intake will be controlled for 10-days (including 3 d prior to immobilization and 7 d of immobilization) providing an energy balanced diet containing 1 weight per day equally distributed over breakfast, lunch, and dinner (15). Participants will be instructed to refrain from vigorous-intensity physical activity (running, fast cycling, competitive sports, carrying heavy loads, etc.) and refrain from alcohol consumption for 3 d prior to and during the 7-day single leg immobilization. Daily activity will be monitored with a SenseWear Armband, which subjects will be required to wear from t=-3 d until the end of the experimental trial.

Participants will have one leg immobilized by means of knee bracing (http://www.breg.com/products/knee-bracing/post-op) thus the contralateral limb will act as an internal control. The leg to be immobilized will be chosen in a random counterbalanced manner to result in equal numbers of participants having their weaker or stronger legs being immobilized. The brace (http://www.breg.com/products/knee-bracing/post-op) will be worn in a fixed flexion position at 140° (i.e., 40° from full extension) with a plastic band that is removed to check for pressure points daily and resealed with a custom-modified plastic strap that is melted to seal the strap, as we have done previously19,20. The reason for this is that the brace cannot be removed without breaking the plastic seal, but could be broken in the case of an emergency and the brace removed entirely, if required. Using this model of disuse we have found the immobilizing brace to be well tolerated, not restrictive, and importantly it does not occlude for popliteal and/or femoral artery blood flow, and we have had no cases of edema or venous thrombosis in previous studies. Participants will also be provided with elastic support bandages to lessen the risk of deep vein thrombosis.

On t=0 d, a blood sample, saliva sample and muscle biopsy will be collected for the assessment of muscle protein synthesis rates. Muscle strength and volume will be assessed using a Biodex and ultrasonography respectively. A knee brace will be placed to start the 7-day single leg immobilization phase. The choice of leg for immobilization will be randomized and balanced for dominance according to maximal isometric strength. At the end of the immobilization phase (t=7 d), a blood sample will be obtained, muscle biopsies will be collected from both the immobilized and non-immobilized leg, and muscle volume and strength will be assessed. After strength is assessed in both legs, a further muscle biopsy will be taken to assess the effect of 17β-estradiol on muscle response to a single bout of exercise (Biodex) in both the non-immobilized and immobilized leg 3-hrs after the exercise bout.