Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans

Abstract

Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents (e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysterone-containing products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohibited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected in vitro in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed. These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 “other anabolic agents”.

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Archives of Toxicology
https://doi.org/10.1007/s00204-019-02490-x
REGULATORY TOXICOLOGY
Ecdysteroids asnon‑conventional anabolic agent: performance
enhancement byecdysterone supplementation inhumans
EduardIsenmann1,2· GabriellaAmbrosio3· JanFelixJoseph3,4· MonicaMazzarino5· XavierdelaTorre5·
PhilippZimmer1,6· RymantasKazlauskas7· CatrinGoebel7· FrancescoBotrè5,8· PatrickDiel1· MariaKristinaParr3
Received: 8 March 2019 / Accepted: 16 May 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance
physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents
(e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However,
scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysterone-
containing products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was
carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the
performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance
enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohib-
ited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for
the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were
observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected invitro
in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in
one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed.
These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results
strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 “other
anabolic agents”.
Keywords Sports performance· Doping· Ecdysterone· Spinach extract· Humans· Resistance training
Eduard Isenmann and Gabriella Ambrosio contributed equally.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0020 4-019-02490 -x) contains
supplementary material, which is available to authorized users.
* Maria Kristina Parr
maria.parr@fu-berlin.de
1 Department forMolecular andCellular Sports Medicine,
Institute forCardiovascular Research andSports Medicine,
German Sport University Cologne, Cologne, Germany
2 Department ofFitness andHealth, IST University ofApplied
Sciences, Duesseldorf, Germany
3 Institute ofPharmacy, Pharmaceutical andMedicinal
Chemistry (Pharmaceutical Analysis), Freie Universitaet
Berlin, Koenigin-Luise-Str. 2 + 4, 14195Berlin, Germany
4 CoreFacility BioSupraMol, Department ofBiology,
Chemistry, Pharmacy, Freie Universitaet Berlin, Berlin,
Germany
5 Laboratorio Antidoping FMSI, Rome, Italy
6 Division ofPhysical Activity, Prevention andCancer,
German Cancer Research Center (DKFZ) andNational
Center forTumor Diseases (NCT), Heidelberg, Germany
7 Australian Sports Drug Testing Laboratory, National
Measurement Institute, NorthRyde, NSW, Australia
8 Department ofExperimental Medicine, “Sapienza”
University ofRome, Rome, Italy

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Introduction
Ecdysteroids are widely marketed to athletes as dietary
supplements advertising to increase strength and muscle
mass during resistance training, to reduce fatigue and to
ease recovery. Several studies have reported a wide range
of pharmacological effects of ecdysteroids in mammals,
most of them beneficial to the organism. The most active
phytoecdysteroid, ecdysterone (a “Russian secret”, chemi-
cal structure in Fig.1), was already suspected to be used
by Olympic athletes since the 1980s. At present, increasing
numbers of dietary supplements containing ecdysteroids
are marketed as “natural anabolic agents”. Their advertis-
ings promise to increase strength and muscle mass during
resistance training, to reduce fatigue, and to ease recovery.
Extensive investigations on the possible growth-promoting
effects of ecdysterone in various animal species (rats, mice,
Japanese quail and cattle) and a few in humans were reported
(Bathori etal. 2008; Courtheyn etal. 2002; Dinan 2001,
2009; Dinan and Lafont 2006; Gorelick-Feldman etal. 2008;
Haupt etal. 2012; Kumpun etal. 2011; Lafont and Dinan
2003; M McBride 2013; Parr etal. 2014; Slama and Kod-
koua 1975; Slama etal. 1996; Tchoukouegno Ngueu 2013;
Toth etal. 2008; Wilborn etal. 2006). Stimulation of pro-
tein synthesis was already reported in the 1960 (Arking and
Shaaya 1969; Burdette and Coda 1963; Okui etal. 1968)
and Bathori etal. (Bathori etal. 2008) reported its anabolic
effect in humans.
Conversely to anabolic–androgenic steroids (AAS) that
increase muscle mass mainly through their binding to andro-
gen receptor (AR), no nuclear receptor that is homologous
to the ecdysone nuclear receptor (EcR) found in insects has
yet been described in mammals (Gorelick-Feldman etal.
2008). Ecdysterone has been characterized as devoid of
binding ability to either AR, estrogen receptor (ER, where
ERalpha was targeted), or glucocorticoid receptor (Bathori
etal. 2008; Seidlova-Wuttke etal. 2010). However, only
recently, binding of ecdysterone to the ERbeta could be
shown invitro and in silico (Parr etal. 2013, 2014, 2015b).
An effect even exceeding that of the AAS metandienone
was found invitro (Parr etal. 2015a) and Chermnykh etal.
reported that ecdysterone showed an anabolic effect stronger
than that of metandienone already without combination with
training while metandienone, in contrast, showed no effect if
not combined with training (Chermnykh etal. 1988). Doses
higher than 5μg/kg body weight (BW) were reported as
active while lower doses did not result in anabolic activities
(Bathori etal. 2008; Chermnykh etal. 1988; Wilborn etal.
2006). Even if there are lots of rumors on ecdysterone mis-
use by athletes, only few scientific studies are available to
demonstrate its performance-enhancing effect. After 20days
of supplementation, Azizov reported a significant increase
in running capacity of mice. In forced swimming tests, they
reported that rats supplemented with ecdysterone were
able to swim significantly longer than the control animals
(Azizov and Seifulla 1998). An increased grip strength in
rats was also reported and a phosphatidylinositol-3-phos-
phate kinase (PI3K)-mediated mechanism is discussed
(Gorelick-Feldman etal. 2008).
In contrast to cell culture and animal studies, ecdysterone
supplementation to improve performance has not yet been
extensively investigated in humans. Apart from the working
group around Wilborn (Wilborn etal. 2006), no detailed
investigation in humans has yet been carried out. Therefore,
in our study, we report on the evaluation of the effect of
a long-term administration of an ecdysterone-containing
dietary supplement with a special focus on the increase in
performance during resistance training in humans.
Serum and urine samples were analyzed for endogenous
hormones and liver enzymes. Furthermore, a complete
anti-doping screening was carried out to exclude under-
lying effects from potential cross contaminations in the
supplement.
Methods
Training investigation (in vivo)
Subjects
Forty-six voluntary healthy male subjects (n = 46) took par t
in the investigation successfully. Six subjects were noticed
as drop out due to missing at more than ten percent of all
training sessions in the study period or personal reasons.
At the beginning, they were 25.6years (SD 3.7 years),
181.9cm (SD 6.3cm) tall and weighed 80.0kg (SD
9.1kg). All subjects provided a 1-year barbell training
experience and ability to perform basic strength exercises
as back squat, deadlift, and bench press. For inclusion
Fig. 1 Chemical structure of ecdysterone

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in the study, all subjects were non-smokers, did not take
any medication or other dietary supplements, and were
injury-free for at least half a year.
This study was approved by the Ethics Committee of
the German Sport University Cologne and carried out
on the basis of the Helsinki agreement in double-blind
design. All participants provided written informed con-
sent prior to their participation. They were assigned to
four different groups (matched according to performance
and body composition): placebo group (PL, n = 12),
ecdysterone1 group (Ec1, n = 12), ecdysterone2 group
(Ec2, n = 10), and control group (CO, n = 12).
Supplements anddosage
As source of ecdysterone, the dietary supplement “Peak
Ecdysone” (PeakPerformance Products SA, Roodt-sur-
Syre, Luxemburg) was used. The product is labelled to
contain 100mg of ecdysterone from spinach extract plus
100mg of leucine.
The volunteers out of the Ec1 group took two cap-
sules of “Peak Ecdysone” per day as recommended on the
label of the product. The Ec2 group took a high dosage
of ecdysterone (eight capsules of “Peak Ecdysone” each
day) over the entire intervention period. The PL group
took two placebo capsules each day over the same period.
The CO group took only two capsules of “Peak Ecdysone”
without training. Each group took half of their nutritional
supplementation dose in the morning after breakfast and
the other half on training days immediately after training
or on non-training days in the evening.
Training system
The PL, Ec1, and Ec2 participated in a 10-week resistance-
training program with three training sessions a week and a
two split training plan (details as supplemental material).
Each training plan consists of six barbell exercises for the
whole body. Every training day was followed by a resting
day. In weeks 1–6, the subjects performed three sets of 12
repetitions for each exercise. After week 6, they performed
only three sets with eight repetitions. Participants increased
their training weight (2.5–5kg) under the supervision of
the supervisor every week, except in weeks four and seven
(recovery weeks), starting with an intensity of 70% of their
one-repeat ability (1-RM). The technically correct execution
of the respective exercises was always in focus. If this could
not be guaranteed, the weight was reduced or not increased
according to plan. Before (t1) and after (t2), the training
period medical examination as well as performance evalu-
ations were carried out. Between the tests and the training
intervention, there was a 72–96h break. Endocrinological
and compliance parameters (anti-doping screening) were
also checked after 5 weeks (thalf). The complete examina-
tion procedure is shown in Fig.2.
Outcome measures
Anthropometric parameters
In the pre- (t1) and post-test (t2) medical examination,
anthropometric parameters as weight and height of the par-
ticipants were determined. All subjects were requested to be
sober to the decreases (12h of no food intake). Body compo-
sition (fat-free mass, muscle mass, fat mass, and total body
water) was measured by bio-electrical impedance analysis
using Akern BIA 101 (Akern GmbH, Mainz, Germany).
Fig. 2 Investigation design and procedure

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Test parameters forperformance
After the medical examination, a standardized breakfast
[60g of cornflakes, two bananas, and 300mL of milk (1.5%
fat)] was provided. Afterwards, performance tests were per-
formed as described below. Tests for training effects were
performed as counter movement jump (CMJ, power), one-
repetition (1-RM) back squat (BS, lower body strength), and
1-RM bench press (BP, upper body strength). As measure
for CMJ, the flight time was determined with an optojump
photo cells (Microgate, Bozen, Italy). All subjects had three
attempts with best result being recorded. For the strength
measurements, four warm-up sets were performed (started
with 50% of 1-RM and 10 repetitions followed by weight
increase and number of repetitions reduction), followed by
four maximum force tests. The weight was steadily increased
after a successful test. If a load was not successfully mas-
tered twice in a row, the test was terminated.
Blood andurine samples
Serum and urine samples were collected for further analyses.
Blood serum concentrations of estradiol (E2), testosterone
(Testo), luteinizing hormone (LH), insulin-like growth factor
1 (IGF1), and thyroxin (T4) were determined using specific
immunoassays (ELISAs) at t1 and t2. In addition, serum
concentrations of Testo, LH, and T4 were also determined
at thalf.
To identify potential side effects, clinical parameters for
liver and kidney toxicity were determined in the blood serum
at t1 and t2. Analyzed parameters were creatinine, gluta-
mate–oxaloacetate transaminase (GOT), glutamate–pyru-
vate transaminase (GPT), and gamma-glutamyl transferase
(GGT). Analysis was performed in a laboratory specialized
in clinical routine diagnostics (Labor Dr. Wisplinghoff,
Cologne, Germany).
Urine samples were used for anti-doping screening to
exclude intentional or unintentional co-administration of
prohibited substances in sports. Furthermore, the urinary
profile of endogenous steroids may also provide evidence on
biological influences potentially induced by the administra-
tion of ecdysterone. The analyses were performed in accord-
ance with the procedures used in the WADA-accredited
Anti-Doping Laboratory of Rome, Italy (Laboratorio Anti-
doping FMSI). According to the technical document, the
“steroid profile” is composed of the following analytes (as
free steroids content obtained from the unconjugated steroid
fraction plus those released from the conjugated fraction
after hydrolysis with β-glucuronidase from E.coli): andros-
terone (A), etiocholanolone (Etio), 5α-androstane-3α,17β-
diol (5αAdiol), 5β-androstane-3α,17β-diol (5βAdiol), testos-
terone (Testo), and epitestosterone (EpiT) as well as ratios
of specific steroid pairs, i.e., Testo/EpiT; A/Testo; A/Etio;
5αAdiol/5βAdiol; and 5αAdiol/EpiT. A detailed descrip-
tion of the analytical methods is given in Supplementary
Materials.
Statistical analyses
The current version of Gpower (3.1.9.2, Universitaet Dus-
seldorf, Germany (Faul etal. 2009)) was used for expres-
siveness and power analysis to determine the sample size.
The data collected were used to test for normal distribution
using the Kolmogorov–Smirnov test. Subsequently, with a
2 × 4 Ankova and Bonferroni test time × group effects and
with paired T test, the individual time and group effects were
analyzed. The current version of SPSS (25.0, IBM Statistics,
Armonk, NY, USA) was used. Significant differences are
set at p < 0.05.
Cell culture investigation ofsupplement activity (in
vitro)
For invitro investigation of the supplement activity, a
C2C12 cell line-based assay was used. The standard proto-
col for C2C12 cells was adhered to as in Zheng etal. (Zheng
etal. 2018). This myoblast cell line derived from murine
satellite cells has shown its potential as an invitro model
to study muscle hypertrophy. To obtain the test solution,
4.8mg of the capsule content (according to the labelling
2.4mg of ecdysterone and 2.4mg of l-leucine) were dis-
solved in 10mL of DMSO. This solution is diluted 1:1000
(v:v) prior to the assay.
After 48h treatment of the C2C12-derived myotubes with
solutions of the supplement, the diameters of the myotubes
were determined. Reference solutions of dihydrotestosterone
(DHT), estradiol (E2), and ecdysterone were used as con-
trols. The test procedure was performed as three independent
replicates.
Results
Anthropometrical parameters
After 10 weeks of treatment (training and/or supplement
administration) anthropometrical, medical and performance
parameters were evaluated.
The body weight of pre- and post-tests did not show sig-
nificant differences within all groups (details provided as
in Fig.3, left and in Supplementary Materials). The par-
ticipants from Ec1 and Ec2 increased their body weight
significantly over 10weeks (Ec1 = 2.58kg (SD 1.90kg);
Ec2 = 3.11kg (SD 1.51kg), p < 0.05; *). There is also a sig-
nificant difference in body weight change between Ec2 and
CO (time × group effect, p < 0.05; #). In muscle mass (MM),

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a time effect could also be observed in Ec1 and Ec2 (Fig.3,
right, p < 0.05; *). In change of MM, there was a significant
difference between the PL and Ec2 (time × group effect,
p < 0.05; #). The Ec2 group increased MM more than 2kg
[2.03kg (SD 1.76kg)], while the PL reduced the MM in
average 0.35kg (SD 1.73kg). The Ec1 group also increased
their MM (in average 1.58kg (SD 1.88kg)), but without
significant difference (p = 0.115) to PL. The CO has only a
slight change in the MM of 0.25kg (SD 2.02kg), with no
significant difference to a training group (Fig.3 right).
In fat mass (FM) and total body water (TBW), there was
no significant difference in pre- and post-tests within the
groups and no time × group effect could be observed.
Power andstrength performance
Power performance: counter movement jump (CMJ)
After 10 weeks of nonspecific bounce training, all three
training groups increase their jump height [PL: 1.94cm (SD
1.74cm); Ec1: 2.01cm (SD 1.99cm); Ec2: 2.39cm (SD
1.62cm), details are provided as Supplementary Material].
There are time effects in all three training groups. However,
there is no significant difference in time × group effects in
CMJ.
Strength performance: 1‑RM back squat andbench
press
All three training groups increased their 1-RM back
squat. The PL group improved their squat from 107.5kg
(SD 15.34kg) to 124.17kg (SD 13.59kg), improvement:
16.67kg (SD 4.49kg), 15.5%). Ec1 had an increase of
18.50kg (SD 6.16kg) from 104.17kg (SD 18.58kg) to
122.71kg (SD 17.90kg) (improvement 17.75%) and Ec2
from 100.50kg (SD 11.06kg) to 120.00kg (SD 13.23kg)
(improvement 19.50kg, 19.4%). However, no significant
difference between the groups and no time × group effect
was observed (Fig.4 left). In the second strength perfor-
mance test parameter, the 1-RM BP, it was observed that
all three training groups increased their performance sig-
nificantly. The PL group had an increase of 3.33kg (SD
Fig. 3 Individual changes in body weight (in kg, left) and of muscle mass (in kg, right) at pre- and post-intervention test, * indicates time effect;
# represents group × time effect (both p < 0.05)
Fig. 4 Results of strength tests performed pre- and post-intervention, 1-RM back squat (left), 1-RM bench press (right), * indicates time effect; #
represents group × time effect (both p < 0.05)

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3.74kg) (relative 3.59%) from 92.71kg (SD 13.46kg) to
96.04kg (SD 11.15kg). In contrast, both supplement groups
increased their 1-RM bench press more than 8kg. In Ec1
an increase from 82.92kg (SD 15.73kg) to 92.50kg (SD
13.73kg), improvement 9.58kg (SD 2.79kg), 11.5%) and in
Ec2 from 88.75kg (SD 13.08kg) to 97.25kg (SD 10.30kg),
improvement: 8.50kg (SD 4.44kg), 9.5%) was observed
(Fig.4 right).
Supplement analyses
The extraction procedure for ecdysterone resulted in an
amount of 6mg ecdysterone per capsule for this product.
In addition, the supplement was checked for the absence
of contamination with other performance-enhancing drugs.
No contamination of these products with substances that are
prohibited in sports was found (Supplementary Material).
Serum sample analyses
Serum concentration ofecdysterone inblood sample
Serum concentrations of ecdysterone were determined in
the groups and results are displayed in detail in Fig.5. As
expected, concentrations increased with time. Furthermore,
dose-dependent values were detected, i.e., the highest ecdys-
terone concentration was obtained in Ec2 group, where vol-
unteers took the highest dose (8 capsules) of supplement.
Baseline values of ecdysterone (concentrations close or even
below LOQ) were seen prior to administration (t1) and in
the PL group.
Endocrine hormone analysis
To investigate potential effects of ecdysterone, training
and combinations thereof on the endocrine system blood
serum concentrations of E2, Testo, LH, IGF1, and T4 were
determined at t1 and at t2. In addition, Testo, LH, and T4
serum concentrations were also determined at thalf.
The average change of the respective serum hormone
concentrations between t1 and t2, normalized for individual
serum concentrations of the participants at t1, is shown in
Fig.6.
No changes in serum Testo and LH were seen. How-
ever, in IGF1 serum concentrations, a significantly differ-
ent pattern of change compared to the placebo group (time
effect, p < 0.05; * and time × group effect p < 0.05; #) was
observed. While training resulted in a decrease of IGF1 for
the placebo group at thalf, treatment with ecdysterone could
antagonize this. In E2 each group decreased its concentra-
tion, but only in Ec1, a time effect was observed (p < 0.05;
*).
The decrease of T4 concentrations induced by training
was the same in the ecdysterone groups compared to the
placebo group (time effect, p < 0.05; *). The comparison
with the concentrations determined at thalf confirmed this.
Five-week uptake of ecdysterone resulted in a significant
change in the T4 serum concentrations compared to the
control group (p < 0.05; #) (Supplementary Material). After
10 weeks, there was no significant difference between any
group any more.
Testo and LH serum concentrations were also determined
at thalf (Supplementary Material). In Testo, each group
decreased their concentrations. In PL, Ec2, and CO, a time
effect is also observed (p < 0.05; *) Despite significant dif-
ferences in various endocrinological parameters at different
times, no change can be attributed exclusively to ecdysterone
supplementation.
Biomarker analysis forside eects
Serum concentrations of the biomarkers of liver and kid-
ney toxicity [creatinine, glutamate–oxaloacetate transami-
nase (GOT), glutamate–pyruvate transaminase (GPT), and
Fig. 5 Serum concentrations of
ecdysterone ([ng/mL of serum],
grouped boxpots) in the differ-
ent treatment groups at t1, thalf,
and t2, one outlier (Co group)
eliminated

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gamma-glutamyl transferase (GGT)] did not change signifi-
cantly over the 10-week intervention period for all groups.
Urine sample analyses
No hint on significant alteration of the steroid profile after
administration was detected. No other performance-enhanc-
ing drugs or significant alterations in the urinary steroid pro-
file were detected in anti-doping screening.
In vitro investigation ofthesupplement
C2C12 cells, a myoblast cell line derived from murine
satellite cells, have been used as an invitro model to study
muscle hypertrophy through ecdysterone. After 48h of
treatment, the diameters of myotubes were determined.
The results are compared with different controls (Supple-
mentary Material). The results show an anabolic activity
of the supplement extract (Ecdy cap) invitro. Increased
diameters of C2C12 derived myotubes were detected,
which were significantly different from the control
(time*group effect, p < 0.05; #). Hypertrophy was found
similar to that obtained by dihydrotestosterone (DHT),
estrogen (E2), or pure ecdysterone reference (Ecdy lab).
There are no differences within the treatment with the dif-
ferent hormones.
Fig. 6 Average change of the respective hormone serum concentra-
tions between T1 and T3, setting the individual serum concentrations
of the participants, normalized for individual serum concentrations of
the participants at T1. * Indicates time effect; # represents group ×
time effect (both p < 0.05)

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Discussion
Our data showed positive effects of ecdysterone on
anthropometric (BW and MM) and performance param-
eters (1-RM BP). These results could be confirmed by the
invitro study. In addition, a dose-dependent effect could
also be observed in various parameters (BW, MM, and
serum concentration of ecdysterone). Furthermore, side
effects that are explicitly attributable to ecdysterone sup-
plementation could not be demonstrated. Negative effects
on creatinine, GOT, GPT, or GGT could not be observed.
Similarly, no significant alteration of urinary steroid
hormones was detected. This suggests that the anabolic
effect of ecdysterone is based on a mechanism that is dif-
ferent than that of testosterone, DHT, and synthetic AAS.
Furthermore, this is also in line with no pseudo-endoge-
nous steroid administration, either as non-compliance to
the study protocol or due to a cross contamination of the
supplement.
Anthropometric andperformance parameter
The positive effect of ecdysterone administration and
training on body weight (BW) and muscle mass (MM)
could be clearly shown (Fig.3). In both parameters, a posi-
tive time effect was generated in Ec1 and Ec2. In addi-
tion, a dose-dependent effect could be observed. Ec2 (high
dose) additionally showed significant time × group dif-
ferences to CO and PL. Similar positive effects on body
weight and muscle hypertrophy have been demonstrated
in 600mg testosterone administration (Bhasin etal. 1996).
Although the effects are not as strong as with testoster-
one supplementation (in human), significant differences
between PL, Ec1, Ec2, and CO can be detected. These
results are corroborated by the invitro study (hypertrophy
in C2C12 myotubes). The anabolic effects of the Ecdy
cap are similar to the hypertrophic effects of DHT, E2,
and Ecdy lab (Supplementary Material). Similar ana-
bolic effects invitro have been reported earlier by differ-
ent groups (Parr etal. 2014, 2015a; Zheng etal. 2018).
In addition, the positive effect of ecdysterone in various
animal experiments was also demonstrated earlier as well
(Bathori etal. 2008; Courtheyn etal. 2002; Dinan 2001,
2009; Dinan and Lafont 2006; Gorelick-Feldman etal.
2008; Parr etal. Parr etal. 2014, 2015a). As a result, it
can be concluded that ecdysterone has a positive anabolic
effect on muscle growth in humans, similar to cell culture
and animal studies.
In performance, all three training groups increased
their power (CMJ) and strength (1-RM BS, 1-RM BP)
performance significantly. In CMJ, the improvement of
the training groups was most likely due to motoric learn-
ing. Thus, the increased leg strength may have caused the
increase in performance (Wisløff etal. 2004). However,
on average, the jump performance is not fully developed,
so that neural adaptations are more likely to improve the
jump performance. For the training intervention, a lin-
ear periodization model was used, which systematically
increases the weight to maximize strength. In 1-RM BS,
the improvement in each group is similar to the investiga-
tion of Schoenfeld etal. (Schoenfeld etal. 2015) and Joao
etal. (Joao etal. 2014), who also used a linear periodiza-
tion model. This means that the conceptual training design
achieves similar results as the previous studies. Compared
to the strength development during testosterone supple-
mentation and strength training (Bhasin etal. 1996), there
was no significantdifference between the placebo and the
two ecdysterone groups. However, there is a tendency for
an increase in performance by the supplementation of
ecdysterone as well as a dose-dependent effect (Fig.4 left).
This can be confirmed by the enhancement of the aver-
age training weights in the squat of the individual training
groups (Supplementary Material). In upper body strength
development, significant differences between the groups
were observed. Both ecdysterone groups increased their
performance significantly compared to the placebo group
(Fig.4 right). These results are in contrast with Wilborn
(Wilborn etal. 2006), who found no differences between
ecdysterone and the placebo group. The different observa-
tions can be attributed to different training systems as well
as ecdysterone concentrations. Wilborn etal. did not use a
systematic linear training model, but a wave-shaped train-
ing model with individual increase and a different ecdys-
terone concentration. Both factors play a decisive role,
which can lead to different observations. In this study, it
can be clearly recognized that ecdysterone has a positive
effect on upper body performance.
Supplement analyses
The quantification of ecdysterone in the supplements revealed
an amount of 6mg per capsule, which is considerably lower
than the amount labelled on the bottle (i.e. 100mg per cap-
sule). Thus, a daily dose of 12mg of ecdysterone, i.e. 0.15mg/
kg BW in an 80-kg volunteer, was administered in Ec1 and
CO, while the high-dose group (Ec2) received a daily dose of
48mg of ecdysterone or 0.6mg/kg BW in an 80-kg volunteer.
In agreement to this, a dose-dependent increase of ecdysterone
serum concentrations after ingestion (Fig.5) and bioactivity
of the supplement extract in the invitro assay were detected
(Supplementary Material). The label of the supplement also
indicated leucine as ingredient (100mg/capsule). Effects of
leucine administration on skeletal muscle performance have
been reported. However, in these studies, the daily uptake

Archives of Toxicology
1 3
of leucine was in the range of 80mg/kg bodyweight, which
would mean 6.4g in a 80kg person (Borack and Volpi 2016;
Gnanou etal. 2006). Therefore, we can exclude that the small
daily dose of leucine provided via the capsules may have any
relevance for the observed physiological effects in this study.
Serum parameters
Serum concentration ofecdysterone
The determination of ecdysterone in blood serum resulted
in increasing concentrations in all supplementation groups
(Ec1, Ec2, and CO) during the study. A clear dose-dependent
increase in ecdysterone is detected; thus, concentrations in
Ec2 group (administration of 8 capsules) were considerably
higher than in Ec1 and CO group (both administration of 2
capsules). Variances in concentrations in all groups most likely
result from different individual pharmacokinetic parameters
and slightly different time intervals, since last supplement
administration, even if all volunteers administered the last cap-
sule in the morning when also sample collection took place.
Detectable baseline concentrations of ecdysterone (most of
them close to the lower limit of quantification) at t1 as well as
in thalf and t2 of the placebo group (PL) most likely resulted
from regular diet.
Endocrine hormone parameters
During the 10-week intervention study, various hormonal
changes were observed. However, no change can be exclu-
sively related to the supplementation of ecdysterone (Fig.6
and Supplementary Material). Possible tendencies and posi-
tive effects on IGF1 could possibly be explained by intensive
training. Intensive training may negatively affect IGF1, which
could be counteracted by supplementation with ecdysterone
(Fig.6). However, further investigations will be needed to con-
firm this assumption.
Furthermore, an influence of the intensive training on T4
could be determined. There are no differences between the
three training groups (PL, Ec1, and Ec2).
For Testo, E2, and LH, no explicit change by supplemen-
tation and/or training was observed. This corroborates the
assumption that ecdysterone has no direct influence on expres-
sion of Testo, E2, or LH. In summary, further investigations
are, therefore, necessary to obtain more detailed information
on the influence of ecdysterone on hormone expression.
Conclusion
This project demonstrates the performance-enhancing effect
of ecdysterone in humans. Thus, our results strongly suggest
including ecdysterone in class S1 “Anabolic Agents”. As it is
reported in the literature, the mechanism of action of ecdys-
terone appears as independent from the androgen receptor
activation, but it is rather exhibited by the activation of the
estrogen receptor beta. However, further investigations on
the activity of ecdysterone are recommended. They should
also include a controlled administration trial of ecdysterone
in humans to elucidate the metabolism of ecdysterone and
to evaluate possibilities for its improved detection in doping
control analyses.
Acknowledgements The authors acknowledge the financial support
from the World Anti-Doping Agency (grant no. WADA 15C18MP).
Dr. Jan F Joseph, Core Facility BioSupramol, Freie Universitaet Berlin
(FUB), Germany, is acknowledged for analytical support and Steffen
Loke, Institute of Pharmacy, FUB, for copyediting.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
References
Arking R, Shaaya E (1969) Effect of ecdysone on protein synthesis in
the larval fat body of Calliphora. J Insect Physiol 15(2):287–296
Azizov AP, Seifulla RD (1998) The effect of elton, leveton, fitoton
and adapton on the work capacity of experimental animals.
Eksperimental’naia i klinicheskaia farmakologiia 61(3):61–63
Bathori M, Toth N, Hunyadi A, Marki A, Zador E (2008) Phytoecdys-
teroids and anabolic-androgenic steroids–structure and effects on
humans. Curr Med Chem 15(1):75–91
Bhasin S, Storer TW, Berman N etal (1996) The effects of supraphysi-
ologic doses of testosterone on muscle size and strength in normal
men. N Engl J Med 335(1):1–7. https ://doi.org/10.1056/NEJM1
99607 04335 0101
Borack MS, Volpi E (2016) Efficacy and safety of leucine supplemen-
tation in the elderly. J Nutr 146(12):2625S–2629S. https ://doi.
org/10.3945/jn.116.23077 1
Burdette W, Coda R (1963) Effect of ecdysone on the incorporation
of 14C-Leucine into hepatic protein invitro. Proc Soc Exp Biol
Med 112:216–217
Chermnykh NS, Shimanovskii NL, Shutko GV, Syrov VN (1988) The
action of methandrostenolone and ecdysterone on the physical
endurance of animals and on protein metabolism in the skeletal
muscles. Farmakologiia i toksikologiia 51(6):57–60
Courtheyn D, Le Bizec B, Brambilla G etal (2002) Recent develop-
ments in the use and abuse of growth promoters. Anal Chim Acta
473(1–2):71–82. https ://doi.org/10.1016/S0003 -2670(02)00753 -5
Dinan L (2001) Phytoecdysteroids: biological aspects. Phytochemistry
57(3):325–339
Dinan L (2009) The karlson lecture. Phytoecdysteroids what use are
they? Arch Insect Biochem Physiol 72(3):126–141. https ://doi.
org/10.1002/arch.20334
Dinan L, Lafont R (2006) Effects and applications of arthropod steroid
hormones (ecdysteroids) in mammals. J Endocrinol 191(1):1–8.
https ://doi.org/10.1677/joe.1.06900
Faul F, Erdfelder E, Buchner A, Lang A-G (2009) Statistical power
analyses using G* Power 3.1: tests for correlation and regression
analyses. Behav Res Methods 41(4):1149–1160
Gnanou JV, Raj T, Kurpad AV, Regan MM (2006) Branched-chain
amino acid requirements in healthy adult human subjects. J Nutr
136(1):256S–263S. https ://doi.org/10.1093/jn/136.1.256S

Archives of Toxicology
1 3
Gorelick-Feldman J, Maclean D, Ilic N etal (2008) Phytoecdysteroids
increase protein synthesis in skeletal muscle cells. J Agric Food
Chem 56(10):3532–3537. https ://doi.org/10.1021/jf073 059z
Haupt O, Tchoukouegno Ngueu S, Diel P, Parr M (2012) Anabolic
effect of ecdysterone results in hypertrophy of C2C12 myotubes
by an estrogen receptor mediated pathway. In: Schänzer W, Geyer
H, Gotzmann A, Mareck U (eds) Recent advances in dope analy-
sis. Sport und Buch Strauß, Cologne
Joao GA, Evangelista AL, Gomes JH etal (2014) Effect of 16weeks
of periodized resistance training on strength gains of powerlifting
athletes. J Exerc Physiol Online 17(3):102–110
Kumpun S, Girault JP, Dinan L etal (2011) The metabolism of
20-hydroxyecdysone in mice: relevance to pharmacological
effects and gene switch applications of ecdysteroids. Steroid Bio-
chem Mol Biol 126(1–2):1–9. https ://doi.org/10.1016/j.jsbmb
.2011.03.016
Lafont R, Dinan L (2003) Practical uses for ecdysteroids in mammals
including humans: an update. J Insect Sci 3:7
McBride MJ (2013) Phytoecdysteroids: a novel, non-androgenic alter-
native for muscle health and performance. J Steroids Horm Sci
12(01):10–12. https ://doi.org/10.4172/2157-7536.s12-e001
Okui S, Otaka T, Uchiyama M, Takemoto T, Hikino H (1968) Stimula-
tion of protein synthesis in mouse liver by insect-moulting ster-
oids. Chem Pharm Bull 16(2):384–387
Parr MK, Haupt O, Ngueu ST, Fritzemeier K-H, Muhn P, Diel PR
(2013) Estrogen receptor beta mediated anabolic effects—insights
from mechanistic studies on the phytoecdysteroid ecdysterone and
selective ligands. Endocrine reviews 5:6. https ://doi.org/10.1210/
endo-meeti ngs.2013.nrsha .3.sat-340
Parr MK, Zhao P, Haupt O etal (2014) Estrogen receptor beta is
involved in skeletal muscle hypertrophy induced by the phyto-
ecdysteroid ecdysterone. Mol Nutr Food Res 58(9):1861–1872.
https ://doi.org/10.1002/mnfr.20130 0806
Parr MK, Botre F, Nass A, Hengevoss J, Diel P, Wolber G (2015a)
Ecdysteroids: A novel class of anabolic agents? Biol Sport
32(2):169–173. https ://doi.org/10.5604/20831 862.11444 20
Parr MK, Wolber G, Naß A, Ambrosio G, Botrè F, Diel PR (2015b)
ER-beta mediated action of dietary supplement ingredient
edcysterone confirmed by docking experiments. Endocrine Rev
FRI-270
Schoenfeld BJ, Ratamess NA, Peterson MD, Contreras B, Tiryaki-
Sonmez G (2015) Influence of resistance training frequency on
muscular adaptations in well-trained men. J Strength Cond Res
29(7):1821–1829. https ://doi.org/10.1519/JSC.00000 00000 00097
0
Seidlova-Wuttke D, Ehrhardt C, Wuttke W (2010) Metabolic effects
of 20-OH-ecdysone in ovariectomized rats. J Steroid Biochem
Mol Biol 119(3–5):121–126. https ://doi.org/10.1016/j.jsbmb
.2010.01.006
Slama K, Kodkoua M (1975) Insect hormones and bioanalogues:
their effect on respiratory metabolism in Dermestes vulpi-
nus L. (Coleoptera). Biol Bull 148(2):320–332. https ://doi.
org/10.2307/15405 50
Slama K, Koudela K, Tenora J, Mathova A (1996) Insect hormones in
vertebrates: anabolic effects of 20-hydroxyecdysone in Japanese
quail. Experientia 52(7):702–706
Tchoukouegno Ngueu S (2013) Bioactivity of plants secondary metab-
olites: Estrogenic, cytotoxic and anabolic effects on estrogen tar-
get organs of an extract of Erythrina excelsa and Ecdysterone.
German Sport University, Cologne
Toth N, Szabo A, Kacsala P, Heger J, Zador E (2008) 20-Hydroxy-
ecdysone increases fiber size in a muscle-specific fashion in rat.
Phytomedicine 15(9):691–698. https ://doi.org/10.1016/j.phyme
d.2008.04.015
Wilborn CD, Taylor LW, Campbell BI etal (2006) Effects of methoxy-
isoflavone, ecdysterone, and sulfo-polysaccharide supplementa-
tion on training adaptations in resistance-trained males. J Int Soc
Sports Nutr 3:19–27. https ://doi.org/10.1186/1550-2783-3-2-19
Wisløff U, Castagna C, Helgerud J, Jones R, Hoff J (2004) Strong
correlation of maximal squat strength with sprint performance
and vertical jump height in elite soccer players. Br J Sports Med
38(3):285–288
Zheng W, Hemker ML, Xie M, Soukup ST, Diel P (2018) Ana-
bolic activity of a soy extract and three major isoflavones in
C2C12 myotubes. Planta Med 84(14):1022–1029. https ://doi.
org/10.1055/a-0598-4812
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