Estrogen Levels and Leg Muscle Fatigability in Eumenorrheic Young Women After 4 mA Transcranial Direct Current Stimulation
Phase: NA
Conditions: Healthy
Sponsor: University of Iowa

Study Protocol 
April 17, 2023 
IRB approved 07/08/2021. 
NCT 04471805  
  Study Title: 4 mA tDCS, Estrogen, and Leg Muscle Fatigability 
This study employed a double-blind, randomized, crossover design, wherein all subjects received both active 
and sham stimulation during each phase of their menstrual cycle. Each of the 10 subjects completed a total of 
6 visits to the lab. The first visit was a familiarization visit where maximal strength testing was performed to 
confirm leg dominance, and an isokinetic fatigue task (FT) was completed to familiarize each subject with the 
FT performed during the evaluation sessions. Additionally, subjects were asked about the start of their 
previous menstrual cycle, along with the projected start of their next menstrual cycle. The start of the menstrual 
cycle was deemed the first day of menstruation. Sessions were then scheduled to correspond with high and 
low points of estrogen level throughout the cycle. The early follicular phase (days 3–4) was targeted for low 
estrogen levels. The late follicular phase (days 9–10) and mid-luteal phase (days 18–20) were targeted for high 
estrogen levels. Sessions were scheduled so that each subject had both active (4 mA) and sham tDCS during 
each phase (early follicular, late follicular, and mid-luteal) of their menstrual cycle to fully assess the effects of 
stimulation. Due to the diurnal nature of estrogen, visits 2–6 were scheduled at the same time of day for each 
individual subject (i.e., ±2 h for each session). Testing sessions began with a blood draw and assay to assess 
serum estrogen levels, followed by tDCS administration (active or sham), and right and left leg FTs. 
Strength testing was performed on a HUMAC NORM isokinetic dynamometer (CSMi, Stoughton, MA, USA). 
Before beginning the strength testing, subjects completed a warm-up exercise which consisted of 15 
repetitions of knee extension and knee flexion (60◦/s, concentric/concentric). After resting for ≥30 s, three 
repetitions of maximal strength isometric knee extension and flexion were completed at 65◦ and 30◦ , 
respectively, with ≥30 s of rest between each repetition. Then, five repetitions of maximal strength isokinetic 
knee extension and flexion were completed at 60◦/s (concentric/concentric) with ≥30 s of rest between each 
set. The right leg was always tested first, followed by the left. Strong verbal encouragement was provided 
during each repetition to ensure maximal effort from the participants. The greatest torque value acquired during 
either of the strength testing tasks (isometric or isokinetic) was utilized to objectively confirm right-side 
dominance. Strength testing was performed on a HUMAC NORM isokinetic dynamometer (CSMi, Stoughton, 
MA, USA). Before beginning the strength testing, subjects completed a warm-up exercise which consisted of 
15 repetitions of knee extension and knee flexion (60◦/s, concentric/concentric). After resting for ≥30 s, three 
repetitions of maximal strength isometric knee extension and flexion were completed at 65◦ and 30◦ , 
respectively, with ≥30 s of rest between each repetition. Then, five repetitions of maximal strength isokinetic 
knee extension and flexion were completed at 60◦/s (concentric/concentric) with ≥30 s of rest between each 
set. The right leg was always tested first, followed by the left. Strong verbal encouragement was provided 
during each repetition to ensure maximal effort from the participants. The greatest torque value acquired during 
either of the strength testing tasks (isometric or isokinetic) was utilized to objectively confirm right-side 
dominance. 
The isokinetic fatigue task (FT) comprised 40 consecutive repetitions of maximal effort isokinetic knee 
extension and flexion (120◦/s, concentric/concentric). In visits 2–5, a 15-repetition warm-up exercise was 
completed before the FT, as detailed above. The FT was performed on the dominant (right) leg first and ≥5 min 
rest was provided to allow for adequate heart rate and respiratory recovery before starting the left leg FT. 
Vigorous verbal encouragement and visual feedback (i.e., per rep work bars) were given to ensure the 
participants were performing maximal effort contractions throughout the fatigue tasks. The largest torque from 
each repetition was included in the analysis. 
Muscle activity throughout the strength and fatigue tasks was recorded via a wireless EMG system (Ultium- 
EMG, Noraxon, USA Inc., Scottsdale, AZ, USA). EMG electrodes (3M Red Dot Monitoring Electrode, Model 
2560; 3M Corp., St. Paul, MN, USA; 2 cm between each 1.3 cm effective area) were secured bilaterally over 
the rectus femoris, vastus medialis, vastus lateralis, and semitendinosus corresponding to a 3D muscle map 
which followed SENIAM guidelines and was provided by the EMG software (MR 3.14, myoMUSCLE, Noraxon 
USA Inc., Scottsdale, AZ, USA). To increase electrode placement consistency, the same researchers placed 
the electrodes on the subjects during each visit. The electrode sites were prepared by shaving and cleaning 
the electrode site with an alcohol wipe before placing the electrodes. The electrodes and wireless transmitters 

were secured in place with elastic bandages and EMG data were collected at 2000 Hz. 

Sessions 2–6 began with a blood draw in the Clinical Research Unit at the University of Iowa Hospital and 
Clinics (UIHC). Staff nurses collected 4.5 mL blood from the median cubital vein of the left arm (total volume 
collected per subject = 9 mL) for the estrogen assay. Samples were immediately analyzed for serum estrogen 
levels after the blood draws by UIHC Pathology technicians using an Electrochemiluminescence Assay (Roche 
Diagnostics, Basel, Switzerland). 
 
 
Statistical Analysis Plan 
A stimulation condition (active vs. sham) by estrogen level (high vs. low) repeated measures ANOVA was 
performed on the FI values and EMG activity for the right and left extensors and flexors. Post hoc analyses 
(paired t-tests and Bonferroni correction) and effect size (Cohen’s d) were calculated to clarify significant main 
effects and interactions. Significance was accepted at p ≤ 0.05. Normality and sphericity assumptions were 
evaluated with the Shapiro–Wilk test and Mauchly’s test of sphericity for the ANOVAs. Greenhouse–Geisser 
corrections were used when the sphericity assumption was violated. GraphPad Prism 9 (GraphPad Software, 
San Diego, CA, USA) was utilized to perform the analyses.