SHORT-TERM EFFECTS OF BROCCOLI-DERIVED GLUCORAPHANIN ON RECOVERY FROM ECCENTRIC MUSCLE DAMAGE: A DOUBLE-BLIND RANDOMIZED CROSSOVER STUDY
Phase: NA
Conditions: Healthy
Sponsor: Lithuanian Sports University

Study Protocol and Statistical Analysis Plan 
Subjects 
The present study will recruit 17 male volunteers aged 18–35 years who will have no resistance 
training experience in the past 6 months, no arm injuries in the past 2 years, no illnesses in the month 
prior to data collection, and no known allergies to broccoli or other cruciferous vegetables. Because 
no effect-size data from comparable crossover trials will be available (only a parallel-group pilot 
study), we will recruit to a feasibility target of at least ~15 completers and will use a within-subject 
crossover design to limit inter-individual EIMD variability, with post hoc sensitivity analysis (α = 
0.05, 1–β = 0.80) showing we will be able to detect medium Time × Supplement effects (~partial η² 
≥ 0.06). Participants will maintain their regular diet, will abstain from exercise for one week before 
the experiment, and will not be allowed to engage in any additional physical activities during the 
experimental period. Supplement adherence will be quantified via daily logs and will be confirmed 
by sachet count, with compliance expected to exceed 95% for all participants. Participants will be 
surveyed about the consumption of various cruciferous vegetables (cabbage, turnips, mustard greens, 
broccoli, horseradish, arugula, radishes), indicating frequency and approximate amounts, which will 
reveal no significant additional intake. Each participant will read and sign a written informed consent 
form. 
Study Design 
Participants will undergo two separate sessions of muscle-damaging exercise targeting the elbow 
flexors: (1) following seven days of broccoli powder consumption (condition X), and (2) following 
an equivalent placebo regimen (condition Y). The broccoli supplementation will consist of 10 g of 
broccoli powder [a freeze-dried glucoraphanin-rich broccoli powder (99.5% broccoli and 0.5% 
mustard seed)] dissolved in 125 mL of boiling water, providing 320 μg of glucoraphanin per serving. 
It will be consumed when still hot/warm once daily for seven days before and for four days following 
the eccentric exercise protocol. The placebo powder, matched in appearance and preparation, will 
contain no broccoli-derived bioactives. The 7-day, ~320 μg/day glucoraphanin dose (manufacturer 
standard serving) will be selected for feasibility. The selected dose will be higher than those used in 
most studies, as reviewed by Fahey et al. (2015). Data on the complete elimination of sulforaphane 
and its metabolites will range from 12–24 hours to as long as 72 hours. 

Intervention order will be randomized in a double-blind, crossover design with a 14-day washout 
period, during which participants will switch to the alternate supplement and will repeat the same 
protocol using the contralateral arm. Simple randomization will be performed using a random number 
generator for the interventions, with all participants starting the first intervention with their dominant 
arm to ensure equal dominant arm use between the broccoli powder and placebo conditions. Both 
participants and investigators conducting the assessments will be blinded to supplement allocation. 
Supplements will be provided in identical sealed sachets. 
Markers of muscle damage will include reductions in isometric and isokinetic peak torque, restricted 
elbow range of motion (ROM), increased arm girth, soreness, and elevated plasma creatine kinase 
(CK) activity and will serve as dependent variables. On the test day, a fingertip blood sample will be 
collected for CK analysis, followed by assessments of elbow ROM, arm girth, biceps muscle 
soreness, and muscle strength using an isokinetic dynamometer (Biodex Medical Systems, System 4, 
Inc., Shirley, New York, USA). Prior to testing, participants will complete a standardized warm-up 
on an arm ergometer (6 min, workload ≈ body mass in watts). All assessments will be repeated 
immediately post-exercise, and at 48 and 96 h post-exercise. 
In Muscle-damaging Exercise Load 
The subjects will be seated on a Biodex isokinetic dynamometer with the backrest fixed at an angle 
of 90°, and the trunk, pelvis, and upper arm will be stabilized with Velcro straps. The axis of rotation 
will be aligned with the elbow joint, and the lever arm pad will be attached at the wrist. Subjects will 
perform six sets of ten maximal voluntary eccentric contractions of the elbow flexors with an angular 
velocity of 60°/s, starting from 120° elbow flexion to full extension (the full elbow extension angle 
will be considered as 0°), with a 1-min rest between sets. Each subject will use the Biodex 
dynamometer to induce muscle damage in a randomly selected arm, either dominant or non-dominant. 
After 14 days, the same exercise will be repeated with the alternative intervention on the other arm. 
Verbal encouragement will be provided to maintain maximal effort throughout each stretch. Work 
done during each eccentric action will be recorded. 
Strength Assessment 
To evaluate voluntary muscle strength, isometric (PTISOM) and concentric isokinetic peak torque 
(PTISOK) of the elbow flexors will be measured using the same dynamometer (Biodex System 4) 
and the same setup as during the muscle-damaging exercise load. The elbow will be positioned and 

fixed at 90° of flexion, with full elbow extension defined as 0°. PTISOM will be assessed first through 
two maximal voluntary contractions, each lasting approximately 1–2 seconds, with a 1-min rest 
between trials. After a 1-min rest, PTISOK will be measured at an angular velocity of 60°/s through 
a 120° ROM (0°–120°), using two continuous contractions in the same setup. The highest torque 
value from the two trials in each condition will be recorded for analysis. Verbal encouragement and 
real-time visual torque feedback will be provided to promote maximal effort. A decline in voluntary 
contraction force and prolonged recovery will be considered indirect markers of EIMD. 
Range of Motion and Arm Girths 
Arm circumference and elbow joint ROM will be assessed according to standardized protocols. Arm 
girths will be measured using a flexible, non-elastic 1-meter tape to the nearest 0.1 cm, with the 
participant standing upright. Measurements will be taken at the midpoint between the acromion and 
olecranon processes, first with the arm relaxed and then in a fully flexed, contracted state, following 
the recommendations of the American College of Sports Medicine. Elbow ROM will be assessed 
using a digital goniometer (Jamar Plus+ Digital Goniometer, Performance Health, Warrenville, 
Illinois, USA) first with the arm in a neutral resting position. Then, participants will actively move 
their elbow into full flexion and extension, sustaining each position for approximately five seconds 
while the angle will be recorded. 
Musculoskeletal Ultrasound 
Biceps brachii muscle thickness (MT), cross-sectional area (CSA), and distal tendon thickness (TT) 
will be measured using B-mode grayscale ultrasound (10–15 MHz linear-array probe, Echoblaster 
128, Telemed, Vilnius, Lithuania). Imaging procedures will follow the guidelines of the European 
Society of Musculoskeletal Radiology and prior protocols. Participants will sit with the arm supported 
on an armrest and a water-based transmission gel will be applied to minimize transducer pressure. 
MT and CSA will be acquired at two-thirds of the distance between the acromion and antecubital 
crease. Three images per muscle will be captured and analyzed in ImageJ (v.1.46; NIH, USA). For 
TT, the transducer will be aligned with the long axis of the bicep tendon using an oblique ulnar 
approach at the elbow joint. To reduce anisotropy artifacts, participants will slightly bend the arm in 
full supination, and TT will be calculated as the average of measurements taken at 5, 10, and 15 mm 
proximal to the radial tuberosity. Echo intensity (EI) will be analyzed from the same ultrasound 
images using grayscale histogram analysis in ImageJ, with values ranging from 0 (black) to 255 
(white), following similar previous muscle and tendon approaches. Specifically, a region of interest 

will be manually selected to encompass the largest possible portion of the muscle, excluding the 
superficial and deep aponeuroses, and for the tendon, the central region bounded by hyperechoic 
borders. Both assessments will be performed using longitudinal views to ensure consistent anatomical 
alignment across time points. All ultrasound measures will be performed by a single experienced 
operator. Intrarater reliability was assessed prior to data collection on four separate occasions in two 
male participants, showing excellent consistency (MT: ICC = 0.97, CV = 3.46%; TT: ICC = 0.98, 
CV = 2.73%). 
Muscle Soreness 
Muscle soreness will be assessed subjectively using a 10-point visual analogue scale during active 
movement. Each point on the scale will have a written description of soreness: 0 (none), 1 (very 
slight), 2 (slight), 3 (mild), 4 (less than moderate), 5 (moderate), 6 (more than moderate), 7 (intense), 
8 (very intense), 9 (barely tolerable), and 10 (intolerably intense). Subjects will be required to evaluate 
the severity of soreness in their biceps brachii during 2–3 full-range eccentric–concentric 
contractions. Muscle soreness will be widely accepted as an indirect indicator of EIMD. 
Plasma CK Activity 
Approximately 0.25 mL of capillary blood will be drawn from the finger and immediately will be 
centrifuged. The plasma will then be used to measure CK activity using a Spotchem™ biochemical 
analyzer (EZ SP-4430, Menarini Diagnostics, Winnersh-Wokingham, UK) with soft reagent strips 
(Arkray Factory, Inc., Shiga, Japan) reported as micro-Katalytic units per L (µkat·L–1). According 
to the analyzer manual, the normal reference range for human plasma CK using this method will be 
0.9–4.1 µkat·L–1. Elevated plasma CK activity will be considered an indirect biochemical marker of 
muscle membrane damage. 
Statistical Analysis 
Data will be analyzed using SPSS (version 30.0.0; IBM Corp., Armonk, NY, USA) and will be 
presented as mean ± SD. Data normality will be assessed using the Shapiro–Wilk test and visual 
inspection of Q–Q plots, with no clear outliers expected to be identified. A repeated-measures design 
will be used to assess the effects of eccentric EIMD and supplementation on multiple physiological 
outcomes. For all dependent variables except CK and muscle soreness, a three-way repeated-
measures ANOVA will be conducted with Time (Pre, Post, 48 h, 96 h), Supplement (X vs. Y), and 

Arm Condition (eccentrically exercised [E] vs. control [C]) as within-subject factors. For CK and 
muscle soreness, which will only be measured in the exercised arm, a two-way repeated-measures 
ANOVA (Time × Supplement) will be used. Mauchly’s test will be used to assess sphericity, and 
when violated, Greenhouse–Geisser corrections will be applied. Effect sizes will be reported using 
partial eta squared (η²), with values of 0.01, 0.06, and 0.14 interpreted as small, medium, and large 
effects, respectively. Post-hoc pairwise comparisons will be Bonferroni-adjusted. Statistical 
significance will be set at p < 0.05.