Hypothalamic-Pituitary-Adrenal (HPA) Axis Functioning in Overtraining Syndrome: Findings from Endocrine and Metabolic Responses on Overtraining Syndrome (EROS)—EROS-HPA Axis
Cadegiani and Kater Sports Medicine - Open
Hypothalamic-Pituitary-Adrenal (HPA) Axis Functioning in Overtraining Syndrome: Findings from Endocrine and Metabolic Responses on Overtraining Syndrome (EROS)-EROS-HPA Axis
Flavio A. Cadegiani 0
Claudio E. Kater 0
0 Adrenal and Hypertension Unit, Division of Endocrinology and Metabolism, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM/UNIFESP) , R. Pedro de Toledo 781 - 13th floor, São Paulo, SP 04039-032 , Brazil
Background: Overtraining syndrome (OTS) results from excessive training load without adequate recovery and leads to decreased performance and fatigue. The pathophysiology of OTS in athletes is not fully understood, which makes accurate diagnosis difficult. Previous studies indicate that alterations in the hypothalamus-pituitary-adrenal (HPA) axis may be responsible for OTS; however, the data is not conclusive. This study aimed to compare, through gold standard and exercise-independent tests, the response of the HPA axis in OTS-affected athletes (OTS group) to healthy physically active subjects (ATL group) and healthy non-active subjects (NCS group). Methods: Selected subjects were evaluated for cortisol response to a 250-μg cosyntropin stimulation test (CST), cortisol and adrenocorticotropic hormone (ACTH) responses during an insulin tolerance test (ITT), and salivary cortisol rhythm (SCR). Results: A total of 51 subjects were included (OTS, n = 14; ATL, n = 25; and NCS, n = 12). Cortisol response in the CST was similar among the three groups. Conversely, mean cortisol response during an ITT was significantly higher in ATL (21.7 μg/dL; increase = 9.2 μg/dL) compared to OTS (17.9 μg/dL; 6.3 μg/dL) and NCS (16.9 μg/dL; 6.0 μg/dL) (p ≤ 0.001; p = 0.01). Likewise, median ACTH response during an ITT was significantly higher in ATL (91.4 pg/mL; increase = 45.1 pg/mL) compared to OTS (30.3 pg/mL; 9.7 pg/mL) and NCS (51.4 pg/mL; 38.0 pg/mL) (p = 0.006; p = 0.004). For SCR, mean cortisol 30 min after awakening was significantly higher in ATL (500 ng/dL) compared to OTS (323 ng/dL) and NCS (393 ng/dL) (p = 0.004). We identified the following cutoffs that could help exclude or confirm OTS: cortisol level at 30 min after awakening (exclusion = > 530 ng/dL); cortisol response to ITT (exclusion = > 20.5 μg/dL; confirmation = < 17 μg/dL or increase < 9.5 μg/dL); and ACTH response (exclusion = > 106 pg/mL or increase > 70 pg/mL; confirmation = < 35 pg/mL and increase < 14.5 pg/mL). Conclusion: The findings of the present study showed that healthy athletes disclose adaptions to exercises that helped improve sport-specific performance, whereas this sort of hormonal conditioning was at least partially lost in OTS, which may explain the decrease in performance in OTS.
Overtraining syndrome; Sports endocrinology; Hypothalamus-pituitary-adrenal axis; Hormones
The hypothalamus-pituitary-adrenal (HPA) axis
response to ITT are exacerbated in healthy athletes,
compared to sedentary healthy subjects. ITT may
be a tool to evaluate whether the athlete is
wellconditioned and to predict performance, once the
exacerbation of the HPA axis responses may play
an important role in the progressive improvement
in sports performance.
There are intrinsic dysfunctions of the HPA axis
response to a stress situation in OTS-affected athletes,
compared to healthy athletes, in an independent way
from exercise-induced stimulation; the dysfunctions
of the HPA axis are located in the hypothalamus and
the pituitary, and not the adrenals. In case an athlete
is suspected for OTS, an ITT stimulation test may be
performed. In the absence of confounding diseases,
blunted cortisol and ACTH responses most likely
confirm the diagnosis of OTS, with accurate cutoffs.
Two new concepts were unprecedentedly
demonstrated by the study. The first new concept is
that physical activity, at least moderate-to-intense,
elicits conditioning effects of hormonal responses to
stimulation that goes beyond exercise, which we
called as “hormonal conditioning of the athlete”.
Besides helping explain the improvement in the
sports performance, the novel conditioning process
found by our study may be the missing link for the
understanding of the underlying mechanisms of the
improvement observed in several responses to
harmful situations, such as infections, neoplasms,
traumas, inflammations, and psychiatric conditions
that are observed in athletes, and which were not
fully understood so far. The second concept is that
whereas healthy athletes seem to present hormonal
conditioning adaptions, those affected by overtraining
seem to have impaired or maladapted hormonal
conditioning, as over-trained athletes have a blunted
optimized hormone response to stress that seem to be
acquired by athletes; as a sort of deconditioning process,
which indicates that the decreased performance and the
reduced time-to-fatigue observed during OTS; these
two key features of OTS, not yet fully understood, may
be at least partly explained by the present findings.
Despite these unprecedented findings, further studies
are recommended to confirm our results.
The combination of excessive training load and lack of
adequate recovery can lead to a decrease in
sportspecific performance associated with fatigue, termed
overtraining syndrome (OTS). Similar states that are
related to OTS include functional overreaching (FOR)
and non-functional overreaching (NFOR), which differ
in terms of the duration of the signs and symptoms and
in performance after recovery [
Improper recovery, low calorie intake, social problems,
and excessive progression in training volume and intensity
are the key triggers of OTS and correlate states. An
inhospitable environment resulted from the inability to recover
from intense energy demands induced an extensive
dysfunctional adaption (maladaptation) with consequent
abnormal responses of multiple markers [
As OTS and related states can affect 15–60% of elite
], and there are a growing number of high
performance athletes, understanding the pathophysiology of OTS
is essential [
]. As OTS is a diagnosis of exclusion, it is
important to screen for inflammatory, metabolic,
hormonal, psychiatric, and infectious conditions that could be the
primary reason of decrease in sports performance [
Among some proposed biomarkers for diagnosis of
OTS, impaired hormonal responses to stressful tests
induced by maximal exercise have been reported [
although further reproduction of the findings is
recommended. With regard to the likely cause of the impaired
hormonal responses, chronic exposure to stress could
decrease the responsiveness of hypothalamic pituitary
adrenal (HPA) axis [
1, 4, 7–12
]. Other alterations of the
HPA axis were related to fatigue states and could be
helpful for the diagnosis of OTS, such as a decreased cortisol
awakening response (CAR) [
], which is an expected
physiological increase in cortisol levels upon waking, and
altered salivary cortisol rhythm [
]. A decrease in
CAR does not necessarily indicate impaired HPA axis
function, but more likely worsened sleep, which is one of
the features of OTS [
1, 3, 12–30
The impaired hormonal response to exercise-induced
stress reported in previous studies [
] may result from
impaired signaling from the musculoskeletal and
cardiovascular systems to the HPA axis, rather than a decreased
responsiveness of the glands. Moreover, in a recent
systematic review [
], we learned that none of the previous
studies were performed with gold standard functional
tests, which are standardized to identify hormones at the
primary site of the dysfunction caused by OTS (that is, the
changes are not due to a lack of external signaling).
In this study, we performed a complete evaluation of the
HPA axis at different levels by employing gold standard
stimulation tests that are independent of exercises to
ensure the hormone findings are the primary responses. The
present study also aimed to correlate other cortisol findings
] with OTS and to find new criteria for OTS.
A detailed description of the materials and methods and
the raw data can be found elsewhere [
], but the
inclusion and exclusion criteria, study design,
questionnaire, and tests performed in this study are described
Inclusion and Exclusion Criteria
Subjects were recruited through social media (Facebook
and Instagram) and contacted by the main researcher
(FAC) and were required to be male, have present body
mass index (BMI) of 20.0–32.9 kg/m2 (for athletes) and
20.0–30.0 kg/m2 (for non-athletes), and be 18–50 years
old, who do not present previous psychiatric disorders,
do not use any centrally acting drugs, or have not used
any hormonal therapy in the previous 6 months. This
was phase 1 of the inclusion criteria.
A minimum amount of physical activity was required
from athletes, as specified in the EROS study
methodology manuscript [
]. In particular, a minimum of four
sessions and 300 min of moderate-to-intensive training
per week was required, at a training level typical for
professional competition, and at least 6 months of
continuous training. In addition, the subject must be referred to
as an athlete by a professional coach.
Volunteers suspected of OTS were required to disclose
underperformance of at least 10% of reduction of the
previous performance, as confirmed by a sports-related
coach that regularly follows the athlete, unexplained by
conditions that could lead to reduction in performance
including infections (particularly Epstein-Barr),
inflammation, overt hormonal dysfunctions (that would be the
primary cause of the decreased performance), and
psychosocial or psychiatric conditions, whose exclusion of
the confounding conditions was performed by the main
researcher (FAC). For inclusion in the OTS group,
athletes must show persistent fatigue, an increased sense of
effort in trainings that required less effort prior to the
OTS, worsening in sleep quality (this criteria was not
obligatory), and fulfill the recommended protocol for
diagnosis of OTS proposed by the most recent
guidelines on OTS [
Remaining subjects then underwent biochemical
examination (phase 2 of the inclusion process). Subjects
were to show levels within the normal range to be
included in this study, in order to prevent confounding
Design of the Study
All selected subjects signed a written informed consent
for participation in the study, approved by the ethics
committee of the Federal University of São Paulo.
In the EROS-HPA axis arm of the study, we evaluated
peripheral and central components of the HPA axis
(primary or peripheral: adrenal; central: pituitary and
hypothalamus), with cosyntropin stimulation test, that
directly evaluates adrenal responses to synthetic ACTH
], insulin tolerance test (ITT), which evaluates the
integrity of the HPA axis [
], and salivary cortisol
rhythm, to identify patterns of the circadian rhythm of
the cortisol [
After the selection criteria, an initial private interview
about training patterns was performed with the selected
athletes (sedentary subjects were not assessed at this
moment), as part of the EROS study (for all arms). The
interview included questions regarding training intensity
(self-evaluation and evaluation by a professional coach,
on a scale from 0 to 10 compared to athletes of the same
level of training) and time since starting training. Other
questions asked in the interview included whether they
worked or studied besides training, and if they did, how
many hours they worked or studied per day (on average);
self-referred libido (on a scale from 0 to 10 compared to
libido 1 year prior to the questionnaire); details of food
ingestion each day in the 7 days prior to the
questionnaire (including number of calories per kilogram per
day, grams of carbohydrates, proteins and fats per
kilogram per day); and sleeping patterns (average
duration of sleep, self-referred sleep quality on a scale
from 0 to 10, and presence or absence of initial insomnia
and terminal insomnia). For all athletes affected by OTS,
questions regarding the average number of days to
overcome the underperformance state and changes in
sensitivity to heat or to coldness or in sleep quality was
Testing of the HPA Axis
A sequence of tests was then performed in all subjects,
including the cosyntropin stimulation test (CST), the
insulin tolerance test (ITT), and the salivary cortisol
Cosyntropin Stimulation Test (CST)
The cosyntropin (synthetic adrenocorticotropic hormone
[ACTH]) stimulation test is performed with a synthetic
ACTH in high doses (250 μg) in order to directly
stimulate the adrenal glands.
For the CST, blood was collected (time 0) from the
antecubital vein of all subjects at 8.00 AM, after 30 min
of resting and 8 h of fasting, and 250 μg of cosyntropin
was infused. Blood was then collected at 30 min (time 1)
and 60 min (time 2) for analysis of cortisol response
(that is, the cortisol increase in absolute levels [μg/dL] in
response to the infusion of 250 μg of cosyntropin
Insulin Tolerance Test (ITT)
We used a gold standard ITT to evaluate the intrinsic
responsiveness and integrity of the HPA axis, since a
normal response required absence of dysfunctions in all
levels (hypothalamus, pituitary, and adrenals) of the
HPA axis. With a normal CST response, any
abnormality found in the ITT is located either in the
hypothalamus or in the pituitary. Unlike exercise stimulation
tests, whose hormone responses depend on external
neuromuscular and cardiovascular signaling, an impaired
hormone response to ITT means the dysfunction is truly
located in the HPA axis.
Subjects performed the ITT 48 h after the CST,
following the same protocol of fasting, arrival time, and
resting period prior to the beginning of the ITT. Blood
was initially collected (time 0), a dose of 0.1 IU/kg of
regular insulin was infused intravenously and new blood
was collected during hypoglycemia (time 1) and 30 min
after (time 2). After the blood collection during
hypoglycemia, 10 mL of 50% glucose solution was given
intravenously, and high-glycemic index food was offered
ad libitum. At all times, cortisol (μg/dL), ACTH (pg/
mL), and glucose (mg/dL) were assessed, and absolute
ACTH/cortisol ratio was calculated.
Due to the risk of ITT-induced severe hypoglycemia
(unconsciousness), three doses of subcutaneous glucagon
were available (GlucaGen HypoKit, 1 μg, NovoNordisk),
as well as syringes containing 20 mL of 50% glucose
solution and an automated external defibrillator (AED).
Salivary Cortisol Rhythm
After stimulation tests, we assessed salivary cortisol
rhythm (SCR) as an attempt to reproduce previous
findings in fatigue-related states studies [
], as a
potential marker of fatigue.
From 2 to 7 days after ITT, saliva was collected at the
time of awakening, at 30 min after awakening, at 4 PM,
and at 11 PM, and specific recommendations were
provided. Saliva samples were collected by the subjects
themselves, using laboratory kits provided by the
All hormones of the present study (salivary cortisol,
serum cortisol, and serum ACTH) were analyzed by
specific electrochemiluminescence assays using specific
commercial kits at a laboratory, whereas serum glucose
levels were analyzed by an enzymatic assay of
hexokinase. Importantly, the 8 AM serum cortisol was
collected at the same time, while salivary cortisol was
collected at the awakening moment and 30 min later,
regardless if, and for this reason, salivary and serum
cortisol levels are not comparable.
Statistical analysis was performed using IBM SPSS statistics
24 software (IBM, USA). Each parameter was compared
among the three groups (OTS, ATL, and NCS), and
pairwise tests were performed between OTS and ATL, OTS
and NCS, and ATL and NCS, whenever p < 0.05. As none
of the variables depend on other variables, main and
interaction effects were not evaluated (cortisol levels at a certain
time do not reflect the ACTH levels at the same time).
Criteria for normal distribution was evaluated using
Kolgomogorov–Sminorv test. Whenever normal
distribution criteria were met, one-way ANOVA tests were
performed, whereas when results were not normally
distributed, Kruskal-Wallis tests (nonparametric ANOVA
tests) were performed. Adjusted Dunn’s test, Dunnett’s
T3, and Tukey analyses were performed when
differences were statistically significant between the three
groups (p < 0.05), according to the normality criteria.
Baseline Characteristics and Training Patterns of the
From the 146 initially recruited subjects, 51 subjects
were included in the study (34.2%), and divided in three
different groups, of OTS-affected athletes (OTS group;
n = 14), healthy athletes (ATL group; n = 25), and healthy
but physically non-active control subjects (normal
control subjects [NCS]; n = 12). All athletes performed both
resistive and endurance exercises, and all the training
patterns were similar between OTS and ATL. All OTS
were classified as OTS (and not FOR or NFOR) due to
their prolonged recovery time.
Regarding the baseline characteristics, the average age
(OTS = 30.6 years, ATL = 32.7 years, and NCS = 33.2 years)
and average BMI (OTS = 26.7 kg/m2, ATL = 24.9 kg/m2,
and NCS = 33.2 kg/m2) were statistically similar among
the groups. In addition, the average number of minutes of
training per week (OTS = 574.3 min and ATL =
550.0 min), mean training intensity (OTS = 8.79 and ATL
= 8.76, on a scale from 0 to 10), and number of training
days per week (OTS = 5.36 days and ATL = 5.46 days)
were similar between the two groups of athletes.
Basal Hormone Levels
As summarized in Tables 1, 2, and 3, basal serum
hormone levels of OTS are within the normal range, and
similar to ATL and to NCS, as previously observed
1–4, 6–9, 17–29, 34–36
Evaluation of the Adrenal Gland—Cosyntropin
Stimulation Test (CST)
As summarized in Table 1, cortisol response to 250 μg
cosyntropin stimulation disclosed a normal and
comparable responses between OTS, ATL, and NCS. The
normal cortisol responses observed in the OTS-affected
athletes confirm that adrenals are unimpaired in the
Cortisol response to CST (μg/dL)
Evaluation of the HPA Axis—ITT
While basal cortisol levels were similar among the
three groups, cortisol levels were higher in the ATL
during and 30 min after hypoglycemia compared to
the OTS and NCS. The mean cortisol increase was
higher in the ATL compared to the OTS and NCS
(Table 2 and Fig. 1).
At 30 min after hypoglycemia, seven (85.7%) OTS, five
(20%) ATL, and nine (75%) NCS showed cortisol levels
< 18 μg/dL. A cortisol peak of < 17 μg/dL in response to
hypoglycemia was reached by four (28.6%) OTS, none of
ATL, and eight (66.7%) NCS. Cortisol levels of > 20.5 μg/
dL in response to hypoglycemia were found in none of
OTS, 15 (60%) ATL, and three (25%) NCS. A cutoff of
19.1 μg/dL disclosed the highest accuracy (84.6%) to
distinct ATL from OTS, although was not precise for neither
confirmation nor exclusion of OTS. Furthermore, a
cortisol increase of > 9.5 μg/dL was not observed in any OTS,
but was found in 15 (60%) ATL and two (16.7%) NCS. A
cortisol response of < 17.0 μg/dL at 30 min after the peak
of hypoglycemia in the ITT seems to be a reliable cutoff to
help confirm the diagnosis of OTS with a likely high
positive predictive value when evaluated in athletes.
Conversely, a cortisol response of > 20.5 μg/dL and a cortisol
increase of > 9.5 μg/dL during the ITT seems to have a
high negative predictive value (100%) according to our
findings. Whereas ATL displayed a prompt cortisol
response to hypoglycemia, OTS and NCS exhibited a
delayed cortisol response.
Basal ACTH and ACTH during hypoglycemia were
similar between the three groups (Table 3). However,
the late increase of ACTH was higher in the ATL
compared to the OTS (Fig. 2). Meanwhile, the median
ACTH levels 30 min after hypoglycemia in the NCS
were lower than in the ATL and higher than in the
12.1 (± 5.7)
19.7 (± 3.2)
22.9 (± 4.4)
OTS, although the differences were not statistically
significant, whereas ACTH increase was higher in the
ATL than the OTS (Fig. 3).
An ACTH peak of > 46 pg/mL was observed in three
OTS (21.4%), in 18 ATL (72%), and in eight NCS
(66.7%); there is a cluster of blunted ACTH response to
ITT among OTS including 78.6% of subjects. ACTH
were < 35 pg/mL in nine (57.1%) OTS, in three (12%)
ATL, and in four (33.3%) NCS, with an accuracy of 80%
to distinct OTS. Conversely, ACTH peak > 106 pg/mL
was observed in only one OTS (an outlier) whereas it
was observed in eight ATL (32%).
Two (14.3%) OTS, 14 (56%) ATL, and seven (58.3%)
NCS showed a > 34 pg/mL increase in absolute ACTH
levels. Meanwhile, no OTS, 10 (40%) ATL, and three
(25%) NCS showed a > 70 pg/mL increase in ACTH.
The highest accuracy was found for a cutoff of 20 pg/
mL (77.0%), although imprecise to determine the
absence or presence of OTS.
Based on our results, an ACTH response of > 106 pg/
mL seems to be highly predictive of exclusion of OTS,
whereas an ACTH response of < 35 pg/mL increases the
likelihood of the diagnosis of OTS. Despite the highly
variable ACTH increase levels, an increase of > 70 μg/dL
could be useful to exclude OTS, whereas blunted ACTH
increase was only observed in OTS and NCS.
In a subgroup of the ATL, ACTH and cortisol were
also performed 60 min after hypoglycemia, and disclosed
lower levels than 30 min, and therefore were not
measured in the remaining subjects.
During the ITT, ACTH/cortisol ratio was similar
between groups basally and during hypoglycemia, but
was significantly lower in the OTS group compared to
the ATL (p = 0.024) and NCS (p = 0.018) groups
Differences between OTS and ATL: *p < 0.05; **p < 0.01; ***p < 0.005
Differences between ATL and NCS: &p < 0.05; &&&&p < 0.001
SD standard deviation
OTS athletes (OTS)
(n = 14)
11.6 (± 2.5)
12.4* (± 3.3)
17.9*** (± 2.9)
6.3** (± 2.3)
Healthy athletes (ATL)
(n = 25)
12.5 (± 3.1)
15.9& (± 5.3)
21.7&&&& (± 3.1)
9.2& (± 3.7)
Non-active subjects (NCS) (n = 12)
10.9 (± 2.8)
11.8 (± 3.1)
16.9 (± 4.1)
5.9 (± 3.9)
Differences between OTS and ATL: ****p < 0.001
Level of significance = p < 0.05
CI confidence interval; n/s = non significant; n/a = non appliable
Glucose levels were similar initially and during
hypoglycemia between groups, reinforcing the equality
of conditions of the ITT and foreclosing differences due
to the intensity of hypoglycemia.
Salivary Cortisol Rhythm (SCR)
SCR was assessed in 23 of the 25 ATL and in all
OTS and NCS. SCR was similar among groups,
although ATL presents exacerbated and significant
elevation on 30 min after awakening compared to
OTS and NCS (Table 5 and Fig. 4). Conversely, CAR,
awakening, 4 PM, and 11 PM salivary cortisol levels
were similar between OTS, ATL, and SED. Although
CAR was higher in ATL, it did not achieve statistical
A cutoff of 370 ng/dL has a high accuracy (80%) but is
not reliable to exclude or confirm OTS. Conversely,
higher salivary cortisol levels (> 530 ng/dL) were not
observed in any of the OTS and in only one NCS, whereas
they were observed in majority of ATL (12 of 22; 59.1%),
and therefore we suggest that a cutoff of > 530 ng/dL
may be a tool to reinforce the exclusion of OTS
This study examined the role of the HPA axis in OTS.
To learn whether the differences in hormone responses
between OTS and ATL resulted from a physiological
adaptation to sports (which has not been analyzed in
previous studies) or from dysfunctional OTS responses,
we included two control groups, of healthy athletes and
healthy non-active subjects. Overall, we found that OTS
have blunted hormonal responses compared to ATL, but
not to NCS, whereas ATL showed more exacerbated
responses than NCS. It means that athletes affected by
OTS do not disclose actual dysfunctional responses, as
hormonal responses were not different from normal
non-active control subjects, but nullifies the likely
adaptive hormonal responses observed in healthy athletes, as
healthy athletes disclosed different responses compared
to sedentary control subjects, whereas the different
responses compared to NCS were not observed when
compared to OTS-affected subjects. This means that the
different responses, a likely adaptive process observed in
healthy athletes, are nullified when OTS is present.
While CST reflects the adrenal status, ITT discloses
the integrity of the entire HPA axis. As the adrenals
responded normally in the abovementioned ACTH
stimulation test, any abnormality found at the ITT must
be located centrally (either in the hypothalamus or in
the pituitary). Moreover, as the ITT directly stimulates
the glands of the HPA axis (whose pathway of
stimulation is the hypothalamus, then the pituitary, and then
the adrenal glands, in this order), any alterations in
responses due to differences in signaling coming from
the neuromuscular and cardiovascular systems are
abolished. Finally, the response to hypoglycemia does
not depend on sports capacity and conditioning, and
therefore, impaired hormone responses to ITT show a
truly impaired HPA axis.
Indeed, differences in cortisol and ACTH responses
were observed between groups, suggesting differences in
the HPA axis responsivity to stressful situations.
However, instead of the presence of dysfunctional impaired
responses in OTS, exacerbated and hastened responses
were observed in ATL, compared to NCS and OTS, for both
ACTH and cortisol, whereas basal levels were similar among
groups. Basal cortisol and ACTH levels tended to be normal
in OTS, as reported by previous studies [
1, 3, 6, 9–12
although some studies reported either reduced [
increased levels [
]; contrariwise, stimulated cortisol
and ACTH levels showed predominantly reduced
1, 3, 7–9
], which is corroborated by us.
Despite the higher cortisol in the ATL during
hypoglycemia, the valuable point of cortisol is actually
30 min after the hypoglycemia event, due to the delay
between ACTH stimulation and consequent cortisol
release. Cortisol levels were already higher during
hypoglycemia likely to the fact that the reduction in
glucose levels and consequent ACTH stimulation began
minutes before the hypoglycemia event. Although
ACTH was not statistically higher during hypoglycemia
in the ATL, the mean and median of ACTH levels were
substantially increased compared to OTS and NCS. The
reduced ACTH/cortisol ratio observed in the OTS group
30 min after hypoglycemia reflects the blunted ACTH
levels observed in the OTS-affected athletes at this time.
However, a reduced ACTH/cortisol ratio does not
provide additional information in regard to a supposed level
of sensitivity of the adrenal glands to the ACTH
stimulation, as ACTH levels cannot be correlated with the
cortisol levels of the same time [
If we followed the adrenal criteria of < 18 μg/dL as
relative adrenal insufficiency [
], the majority of OTS and
NCS would be considered as affected, although we
modified the ITT protocol after the hypoglycemic episode to
prevent severe hypoglycemia [
]. Importantly, the
relative adrenal insufficiency is only found during a
stimulation test and does not bring about the typical risks of the
classical and frank primary adrenal insufficiency that can
be diagnosed by elevated basal serum ACTH levels and
reduced basal serum cortisol levels .
Differences between OTS and ATL: ***p < 0.005;
Differences between ATL and NCS: &p < 0.05
SD standard deviation
We did not perform the 60 min cortisol after
hypoglycemia as we initially performed with some
athletes (n = 13) and cortisol levels did not disclose
increased responses between 30 and 60 min.
Furthermore, previous studies hypothesized that OTS
was characterized by alterations in the sensitivity of the
HPA axis [
]. Therefore, the ACTH/cortisol ratio
would be altered in OTS-affected athletes. We found the
ACTH/cortisol ratio basally, during hypoglycemia, and
30 min after hypoglycemia was similar among the three
groups at all times. While our results seem to invalidate
the theory regarding the hyposensitivity of the HPA axis
in OTS, an altered ACTH/cortisol ratio does not
indicate a difference in response in the HPA axis, as the
process of releasing ACTH to stimulate cortisol release
can take a few minutes. Therefore, the cortisol level at a
certain time point does not reflect the ACTH level at
that same moment.
The loss of circadian rhythm of the cortisol release
can be a feature of fatigue-related disorders, as
previously described [
], although causality of adrenal
impairment has not been demonstrated. However, an
intact cortisol rhythm was observed in all subjects,
regardless of the group. Nonetheless, an exacerbated
cortisol awakening increase was observed in ATL,
although CAR was not statistically significant due to high
variation of the results. However, if there were three
times more subjects, with the same median and
confidence interval, statistical significance would be achieved
for the CAR. The significant difference was found at
30 min after awakening and seemed to be a reliable
marker of sports conditioning, whose adaption showed
to disappear when OTS is present.
OTS athletes (OTS) (n = 14)
329 (± 222)
Healthy athletes (ATL) (n = 25)
337 (± 131)
Non-active subjects (NCS) (n = 12)
266 (± 149)
324*** (± 116)
166 (± 113)
94 (± 39)
500& (± 168)
144 (± 83)
95 (± 38)
393 (± 149)
130 (± 57)
83 (± 11)
The Hormonal Deconditioning Effect of OTS
It has been previously proposed that hypersensitivity of
the HPA axis (during the beginning of the overtraining
state process) followed by a progression to insensitivity
is involved in OTS [
1, 7, 8
]. However, our results suggest
that the hyposensitive HPA axis is only observed when
compared to healthy athletes. We found the cortisol
response to hypoglycemia was significantly higher among
healthy athletes than OTS and NCS. This shows that the
improvement in HPA axis sensitivity in athletes is loss
during OTS, and not that OTS presents any frank
dysfunctions, as OTS discloses similar responses compared
to the general population (i.e., non-active healthy
subjects with same age, gender, and BMI). Furthermore,
while cortisol response was observed 30 min after
hypoglycemia in OTS, it is unlikely that any further
increased cortisol levels (at any time after 30 min) would
be sustained, as ACTH (the major stimulator of cortisol
release) levels were reduced at 30 min after
hypoglycemia at this group.
In summary, all the findings of the present study,
including cortisol and ACTH responses to ITT and
salivary cortisol 30 min after awakening, seemed to be
positive adaptions to exercises in healthy athletes (in the
absence of OTS) that helped improve sport-specific
performance, and for some reason, this hormonal
conditioning (which were the positive adaptions to exercises)
was at least partially lost in OTS, which may explain the
decrease in performance in OTS.
As healthy athletes disclosed optimized HPA axis
responses to hypoglycemia compared to sedentary
individuals, we suggest that there is a possible hyperresponsivity
of the HPA axis in athletes (as an adaption or conditioning
response) that can be expressed independently of exercise.
Correlations Between Findings with Clinical Features of OTS
Besides fatigue and underperformance, a key feature of OTS
is the inability to sustain a sport-specific performance
capacity throughout an intensive training session [
1–4, 39, 40
OTS-affected athletes are usually able to perform normally
in the beginning of the training load, but are not able to
complete at the expected pace (i.e., OTS-affected subjects
tend to fatigue too early compared to healthy athletes). We
found a similar early increase in ACTH levels among all
three groups, but an early fall in ACTH levels among
OTSaffected athletes, with especially blunted ACTH levels at
30 min after hypoglycemia. These findings (normal early
ACTH increase with late blunted levels) may at least partly
explain the reduced time-to-fatigue found in athletes
affected by OTS. Indeed, ACTH and cortisol are two
fundamental hormones required to sustain performance and pace
in any sport. (Thus, as the integrity of the HPA axis is
required throughout training to maintain performance, a
shortened response duration of the HPA axis in
OTSaffected athletes would affect sustained performance).
Suggestions of Cutoffs for Biochemical Diagnosis of OTS
While the number of subjects included in this study was
small, we were able to suggest helpful cutoffs for the
diagnosis or exclusion of OTS due to a number of
factors. First, the number of subjects of the present study is
significantly higher than previous studies [
the diagnosis of OTS was strictly performed (without
confounding bias), and therefore all 14 OTS presented real
OTS. Finally, the hormone levels were markedly different
between OTS and ATL, with few overlapping results in
most findings. A summary of the possible cutoffs to help
confirm or exclude OTS are described in Table 6.
In summary, to the best of our knowledge, this is the
first study on the endocrine system in OTS to (1)
demonstrate that there are intrinsic dysfunctions of the HPA
axis response to a stress situation in OTS-affected
athletes, compared to healthy athletes; (2) demonstrate
that the hypothalamus and the pituitary, and not the
adrenals, are likely responsible for the changes in the
cortisol response in an independent way from
exerciseinduced stimulation; (3) show that salivary cortisol levels
30 min after awakening are blunted in OTS-affected
subjects, which may be a reliable marker of OTS; (4)
propose useful cutoffs of salivary cortisol, and cortisol
and ACTH responses to ITT, as an additional tool to
help exclude or confirm OTS; (5) demonstrate that
overtrained athletes have a blunted optimized hormone
response to training stress; and (6) indicate that reduced
time-to-fatigue, a key feature of OTS, not yet fully
understood, seems to be at least partly explained by the
The main limitations of the present study are as
follows: (1) the small number of subjects, due to the
excluded subjects when strict criteria was used (but still
larger compared to previous studies) and (2) a high
variability and the lack of normal distribution observed in
ACTH responses, which typically occurs in tests with
ACTH responses, but challenges the statistical
Moreover, the presence of outliers reinforces the
individuality of overtraining syndrome presentation in each
athlete, which leads to distinct responses to different
tests. Also, as all athletes were considered as OTS in
this study (that is, the prolonged overtraining state), it
is unclear whether the changes could be found earlier
in OTS, in the functioning and non-functioning
Whereas healthy athletes seem to present hormonal
conditioning adaptions, those affected by overtraining
Highly predictable of exclusion of OTS (93.9%).
Highly accurate (80%), but unable to help diagnosis OTS
High negative predictive value for OTS (100%)
High positive predictive value for OTS, although not specific (28.6%)
High accuracy (84.6%), but not precise for confirmation or exclusion of OTS
100% specific to exclude OTS
High negative predictive value (92.9%) and highly accurate (80%)
80% accurate to distinguish OTS from ATL
seem to have impaired or maladapted hormonal
conditioning, which may explain the decreased performance
observed during OTS, as a sort of deconditioning
process. Our findings allowed us to suggest useful cutoff
values for the 30 min after awakening cortisol, cortisol
and ACTH 30 min after hypoglycemia, and cortisol and
ACTH increase during the ITT to help exclude or
confirm diagnosis of OTS. Despite these unprecedented
findings, further studies are recommended to confirm
ACTH: Adrenocorticotropic hormone; ATL: Healthy athletes group;
CBG: Cortisol-binding globulin; CST: Cosyntropin stimulation test;
EROS: Endocrine and Metabolic Responses on Overtraining Syndrome;
ITT: Insulin tolerance test; NCS: Normal control subjects; OTS: Overtraining
syndrome; SCR: Salivary cortisol rhythm
We acknowledge the support of the team of Sports Medicine and
Endocrinology Departments of Federal University of São Paulo, who helped
to design the most appropriate methods to assess OTS athletes and to
improve the inclusion and exclusion criteria.
We also acknowledge the DASA Laboratórios da América that provided all
the biochemical material and personal staff to help perform the tests, and
Corpometria Institute, that provided all the body metabolism and
Availability of Data and Materials
DASA laboratórios da América supported with all the biochemical data of
the study, while Corpometria Institute offered all the body composition and
metabolism analysis. CAPES/CNPq and Federal University of São Paulo bases
for access to scientific literature were responsible for providing all the data
for the background and discussion of the study.
FAC performed the research, selection of subjects, performance of the tests,
and analysis of the results and part of the discussions and conclusions. CEK
participated in the design of the study and in the discussions and
conclusions. FAC and CEK performed the discussion, the conclusions, and
the design of the tables. Both authors read and approved the final
Funding was not provided for the study, although the study has been
nonfinancially funded by DASA and by Corpometria Institute.
Ethics Approval and Consent to Participate
The study was approved by the ethics committee of the Federal University
of São Paulo (approval reference number: 1093965), and was performed in
full accordance with the standards of ethics outlined in the Declaration of
Helsinki. All selected participants signed the written informed consent for
participation in the study.
Consent for Publication
We declare that the consent for publication is not applicable to this study.
Flavio A. Cadegiani and Claudio E. Kater declare that they have no
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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