BRIEF RESEARCH REPORT
published: 04 September 2019
doi: 10.3389/fspor.2019.00017
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1
September 2019 | Volume 1 | Article 17
Edited by:
Giuseppe D’Antona,
University of Pavia, Italy
Reviewed by:
Lars R. McNaughton,
Edge Hill University, United Kingdom
Jeremy Coquart,
Université de Rouen, France
*Correspondence:
Chen Fleischmann
chen.ﬂeischmann@sheba.health.gov.il
Specialty section:
This article was submitted to
Exercise Physiology,
a section of the journal
Frontiers in Sports and Active Living
Received: 13 June 2019
Accepted: 16 August 2019
Published: 04 September 2019
Citation:
Fleischmann C, Horowitz M,
Yanovich R, Raz H and Heled Y (2019)
Asthaxanthin Improves Aerobic
Exercise Recovery Without Affecting
Heat Tolerance in Humans.
Front. Sports Act. Living 1:17.
doi: 10.3389/fspor.2019.00017
Asthaxanthin Improves Aerobic
Exercise Recovery Without Affecting
Heat Tolerance in Humans
Chen Fleischmann 1,2,3*, Michal Horowitz 2, Ran Yanovich 1,2,4, Hany Raz 5 and Yuval Heled 2
1 Institute of Military Physiology, IDF Medical Corps, Tel-Hashomer, Israel, 2 Heller Institute of Medical Research, Sheba
Medical Center, Ramat Gan, Israel, 3 Laboratory of Environmental Physiology, Dentistry Faculty, Hebrew University of
Jerusalem, Jerusalem, Israel, 4 The Academic College at Wingate, Wingate Institute, Netanya, Israel, 5 The Faculty of
Agriculture, Food and Environment, Hebrew University, Rechovot, Israel
Objectives: To examine the supplementation effects of the xanthophyll carotenoid
Astaxanthin on physical performance and exertional heat strain in humans.
Design: A randomized double blind placebo controlled trial.
Methods:
Twenty two male participants (Age: 23.14 ± 3.5 y, height: 175 ± 6 cm,
body mass: 69.6 ± 8.7 kg, % body fat: 16.8 ± 3.8) received placebo (PLA, n = 10)
or Astaxanthin (ATX, n = 12) 12 mg/day Per os (P.O), for 30 days, and were tested pre
and post-supplementation with a maximal oxygen uptake (VO2 Max) test and the heat
tolerance test (HTT) (2 h walk at 40◦C, 40% relative humidity (RH), 5 kph, 2% incline).
NIH database registration no. NCT02088242. Gas exchange, Heart rate (HR), Relative
perceived exertion (RPE), and blood lactate were measured during the VO2 Max test.
Heart rate (HR), rectal (Trec), and skin (Tskin) temperatures, RPE, and sweat rate (SR)
were monitored in the HTT. Serum heat shock protein 72 (HSP72), Creatine phospho-
kinase (CPK), C-reactive protein (CRP), and lipid proﬁle were measured before and after
the test.
Results:
The rise in blood lactate caused by the VO2 Max test was signiﬁcantly
diminished in the ATX group (9.4 ± 3.1 and 13.0 ± 3.1 mmole∗l−1 in the ATX and PLA
groups, respectively P < 0.02), as was the change in oxygen uptake during recovery
(−2.02 ± 0.64 and 0.83 ± 0.79% of VO2 Max in the ATX and PLA group, respectively,
p = 0.001). No signiﬁcant differences were observed in the anaerobic threshold or VO2
Max. In the HTT, no signiﬁcant physiological or biochemical differences were observed
(HR <120 bpm, Trec rose by ∼1◦C to <38◦C, no difference in SR).
Conclusions: Astaxanthin supplementation improved exercise recovery. No beneﬁt was
observed for ATX over PLA in response to heat stress. Further examination of Astaxanthin
in higher exertional heat strain is required.
Keywords: astaxanthin, supplementation, exercise nutritional physiology, aerobic exercise, exercise-recovery,
heat tolerance
Fleischmann et al.
Astaxanthin Exercise Recovery and Heat Tolerance
INTRODUCTION
Astaxanthin is a xanthophyll carotenoid food supplement
prevalent in marine organisms (Kidd, 2011). This potent
antioxidant (Kidd, 2011) aﬀects the Insulin\Insulin growth
factor I (IGF1) and the nuclear kinase mitogen and stress-
activated protein kinase-1 (MSK1) signaling pathways, which
were found to be implicated in preconditioning, survival and
longevity in vitro in human keratinocytes (Terazawa et al.,
2012) and in vivo in Caenorhabditis elegans (Yazaki et al., 2011)
Other in vivo experiments in animals have shown Astaxanthin
is associated with reductions in C reactive protein and DNA
damage and improvement of the cell-mediated and humoral
immune responses (Park et al., 2011) and with improvement
in cardiovascular parameters (Fassett and Coombes, 2012). In
exercising mice Astaxanthin induced diminished fatigue, and
reductions in blood lactate, oxidative damage to lipids and
DNA, and muscle injury (Aoi et al., 2003; Ikeuchi et al.,
2006). However, exercise experiments in humans were equivocal,
showing improved endurance as time trial performance in
competitive cyclists (Earnest et al., 2011), vs. no signiﬁcant
improvement in well-trained cyclists (Res et al., 2013) and soccer
players (Djordjevic et al., 2012). During exercise, some evidence
from animal experiments supports enhanced fat utilization over
carbohydrates (Ikeuchi et al., 2006; Aoi et al., 2008), yet no
supplementation eﬀect in endurance exercise and recovery was
established (Brown et al., 2018).
Exertional heat injury is a life threatening condition inﬂicting
many young, healthy individuals, commonly aﬀecting highly
motivated, physically active populations such as military
personnel and athletes (Carter et al., 2005; Casa et al., 2005). Risk
reduction of heat injury includes avoidance of strenuous physical
activity in severe heat load conditions, application of heat
acclimation protocols, and the use of external cooling methods
(Epstein et al., 2000). Preparation for planned military and
athletic activities could potentially enhance resilience to extreme
physical and environmental conditions, reduce the chance of
heat injury and improve injury response and recovery. Several
exogenous agents have been studied either as prophylactic to heat
stress exposure or as post injury treatment yet none were eﬀective
(Moran et al., 1999; Kuennen et al., 2011).
Heat
load
is
a
signiﬁcant
stressor
during
exercise.
Astaxanthin’s activity against stressor induced generation of
reactive oxygen and nitrogen species (RONS) and inﬂammatory
cytokines (Brown et al., 2018) may thus be beneﬁcial in heat
stress conditions. Accordingly, Do et al. demonstrated that
Abbreviations: AT, Anaerobic threshold; ATX, Astaxanthin; BMI, Body mass
index; BSA, Body surface area; CPET, Cardio pulmonary exercise test; CPK,
Creatine phospho kinase; CRP, C- reactive protein; ELISA, Enzyme-Linked
Immunosorbent Assay; FDA, U.S. Food and Drug Administration; GI, Gastro
intestinal; HR, Heart rate; HSP72, Heat shock protein 72; HSR, Heat shock
response; HTT, Heat tolerance test; IGF-1, Insulin-like growth factor 1; MAP,
Mean arterial pressure; MPO, Myeloperoxidase; MSK-1, Mitogen- and stress-
activated protein kinase-1; OBLA, Onset of blood lactate accumulation; OD,
Optical density; PLA, Placebo; PP, Pulse pressure; RER, Respiratory exchange ratio;
RH, relative humidity; RONS, Reactive oxygen and nitrogen species; ROS, Reactive
oxygen species; RPE, Relative perceived exertion; SR, Sweat rate; TCR, Thermal
comfort rate; VO2, Oxygen uptake; VO2 Max, Maximal oxygen uptake.
during development, treatment with Astaxanthin increased
protection of porcine oocytes against heat shock, along with
increased resilience to oxidative stress (Do et al., 2015). In
rodents, Astaxanthin enhanced protection against heat related
damages and oxidative stress (Preuss et al., 2009) and resilience
against heat stress combined with gravitational unloading
(Yoshihara et al., 2018). In a preliminary experiment our
group demonstrated improved heat tolerance with elevated
cardiac tissue concentration of HSP72 protein and HSP70
mRNA in rats (Horowitz M. & Abbas A., unpublished data).
In yellow catﬁsh, Astaxanthin pretreatment improved overall
stress resistance, while elevating hepatic heat shock protein 70
(HSP70) mRNA levels, with increased antioxidant capacity,
and decreased expression of the stress related hormone cortisol
and glucose levels (Liu et al., 2016). Puﬀerﬁsh fed with a diet
containing Astaxanthin produced less reactive oxygen species
(ROS) when exposed to heat stress, and increased production of
superoxide dismutase (SOD), catalase (CAT), and HSP70 mRNA
under high temperature stress, in comparison with the control
(Cheng et al., 2018). Elevation of HSP70 mRNA and HSP72
protein is an important part of the heat shock response (HSR),
representing innate cellular defense mechanisms against heat
related damage (Horowitz, 1998). Overall, across several animal
models, Astaxanthin treatment enhances cellular protection
against heat with corresponding increased levels of HSP70,
possibly by priming key components of the HSR for activation,
and by acting as a potent antioxidant via protection against the
heat stress induced generation of RONS.
Based on the aforementioned knowledge, and in the absence
of known safe substances applicable as preemptive measures
for anticipated heat stress exposure, Astaxanthin emerges as
a potential candidate for enhancing heat resilience through
increased cellular protection, pertinent to both heat and exercise
exposure, as it is a safe food supplement, which may potentially
be consumed chronically without adversely aﬀecting active
populations. Therefore, we set out to determine whether
Astaxanthin supplementation, as a preemptive strategy, could
have an inﬂuence on performance in heat stress combined
with exercise scenarios in humans, and potentially serve as an
added line of defense against heat related injury for individuals
anticipating exposure to heat and exercise. We also chose to
separately evaluate the inﬂuence of Astaxanthin supplementation
on aerobic ﬁtness, since it is a key contributing component to
endurance in the heat (Mclellan et al., 2012) and to determine
whether the potential added cellular protection might inﬂuence
aerobic performance, independently of heat exposure.
The study goals were to determine whether Astaxanthin pre-
supplementation could inﬂuence performance in exercise alone
or in combination with heat stress.
METHODS
In
order
to
evaluate
the
inﬂuence
of
Astaxanthin
supplementation on heat tolerance and on aerobic capacity,
we employed a double blind placebo controlled randomized
trial. The heat tolerance test (HTT), which involves exposure
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Astaxanthin Exercise Recovery and Heat Tolerance
to mild physical activity in controlled heat load conditions
and the maximal oxygen uptake (VO2 Max) test were used
before supplementation and repeated after 1 month of daily
supplementation. The study was approved by the ethical review
boards of the Sheba medical center (reg. no. 1295-13) and of
the IDF Medical Corps (reg. no. 0471-13) and was registered in
the NIH database (reg. no. NCT02088242). Data collection took
place between March of 2015 and March of 2016 at the Heller
institute of medical research located in the Sheba medical center,
Tel-Hashomer, Israel.
Participants
Twenty two young healthy male volunteers, free from illness and
not consuming medications or dietary supplements, completed
their participation in the study after giving their informed
consent and being examined by the study physician. Participants
were interviewed by a nutritionist to ensure an Astaxanthin
free diet and instructed to avoid changing their exercise
routine for the duration of the study, and refrain from
consuming Astaxanthin containing foods, as well as any dietary
supplements for 2 weeks prior to participating in the physical
tests and throughout the duration of the study. They were
randomly assigned to either the supplementation group, who
received 12 mg of Astaxanthin P.O daily as 3 soft gel capsules
of Astapure R⃝ (10% Oleoresin) 4 mg or a placebo identical
in appearance and taste, which contained no Astaxanthin
(Algatech, Ktora, Israel). The Supplement and placebo capsules
were purchased directly from the manufacturer, to guarantee
production of a placebo identical in every way to the supplement,
apart from the presence of the active ingredient. Certiﬁcates
of analysis were issued for each purchased batch, ensuring a
95% purity at least of the active ingredient (Astaxanthin) in the
oleoresin contained in the soft gel capsules.
Treatment
The dose (12 mg) was chosen in accordance with the highest
daily dose approved for human consumption by the U.S. Food
and Drug Administration (FDA) at the time of study approval
and with literature evidence from human experimentation,
demonstrating safety and eﬃcacy at this and higher doses
(Kupcinskas et al., 2008; Yoshida et al., 2010; Choi et al., 2011;
Nakagawa et al., 2011). Supplementation duration (over 30 days)
was chosen in order to ensure adequate time for achieving a
supplemented state and initiating the necessary long term eﬀects,
based on other human experiments involving exercise related
aspects, without any known threat to the subjects’ health and
well-being (Spiller and Dewell, 2003; Bloomer et al., 2005; Earnest
et al., 2011; Miyazawa et al., 2011).
Randomization and assignment to the Astaxanthin or placebo
group was performed by an independent party (the clinical
research division of the Sheba medical center pharmaceutical
services), which also individually dispensed the study product
to the participants. Treatment allocation was disclosed to the
researchers only after study completion. In order to ensure
maximal gastro-intestinal (GI) absorption, participants were
instructed to ingest the supplement or placebo with a meal
containing 15 grams of fat. Supplementation lasted for 30
days, immediately followed by an additional supplementation
period of 5–10 days, during which the physical tests (HTT
and VO2 Max) were repeated, on separate days, in-order to
maintain an eﬀective concentration of the supplement and
ensure the tests were performed under a supplemented state.
Treatment adherence by participants was monitored by keeping
a supplementation log and sending a daily text message after
supplement consumption. A dietary log was also kept for 3 days
before each physical examination day.
Experimental Design and Procedures
Twenty two participants completed the study, after being
randomly assigned to either the Astaxanthin (ATX, n = 12,
age: 22.3 ± 4.0 years) or placebo (PLA, n = 10, age: 24.1 ±
2.60) groups in a double blind manner. Participants in both
groups were of average anthropometrics (Height=173.95 ±
4.0 cm, and 1.75 ± 7.6 cm; Body mass = 68.46 ± 8.0, and
70.96 ± 9.8; BMI = 22.6 ± 2.33, and 23.02 ± 2.40; %body
fat = 13.32 ± 4.15% vs. 17.33 ± 3.41%, in the ATX and PLA
groups, respectively, no signiﬁcant diﬀerence between treatment
groups). Supplementation began after completion of the initial
HTT and VO2 Max tests, and lasted a total of 35–40 days.
The HTT and VO2 Max tests where repeated after 30 days
under ongoing supplementation. Figure 1 is a ﬂow diagram
of the study, detailing the process of participant recruitment,
assignment and testing.
Anthropometric measurement (height, body mass, body fat
from a four points skinfold measurement) was followed by
evaluation of aerobic capacity and heat tolerance which were
conducted on separate days, at least 48 h apart, and followed by
commencement of daily supplementation. Aerobic capacity and
heat tolerance assessment were repeated during the 31–40 day
period of supplementation, while still consuming the supplement
or placebo.
Anthropometry included height (roll-up stadiometer, model
206, Seca medical measuring systems and scales, Germany), body
mass (electronic scales), and determination of body composition
by the four sites (biceps brachii, triceps brachii, suprailiac,
subscapular) skinfold measurement (Lange skinfold caliper, Beta
technology, Santa Cruz, CA) and calculation of fat content and
lean body mass, based on an equation suited to the participant’s
age (Durnin and Womersley, 1974).
The heat tolerance test (HTT) was described by Moran et al.
(2007). Participants were dressed in shorts and tennis shoes and
exposed to 2 h of extreme heat stress (40◦C, 40% RH) in a climatic
chamber, while walking on a motor-driven treadmill (5 kph,
2% incline). Rectal temp. (Trec), skin temp. (Tsk), and heart
rate (HR) were continuously monitored. Fluid consumption
(cold water) was provided ad-libitum from pre-weighed drinking
cans. Trec was measured with a rectal thermistor (YSI-401,
Yellow Springs Incorporated, USA) inserted 10 cm beyond the
anal sphincter. Skin temp. (Tsk) at the chest, upper arm and
calf, was measured using a skin thermistor (YSI-409B, Yellow
Springs Incorporated, USA). Mean Tsk was calculated by Burton’s
equation (Burton, 1935). All temperatures were continuously
recorded (MP150 and Acqknowledge software, version 3.9,
Biopac systems, USA). Heart rate (HR) was continuously
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Astaxanthin Exercise Recovery and Heat Tolerance
FIGURE 1 | Study ﬂow diagram.
monitored by a heart rate monitor (model: RS800CX, POLAR,
Finland). Blood pressure (BP) was monitored at pre-set time
points (before the test, after 1 h of walking, at the end of
the test, and every 15 min during recovery, for 1 h after the
test) using an automated blood pressure monitor (Omron
m6 comfort, Omron healthcare, Japan). Fluid balance was
determined from nude body mass, measured before and after
each trial, adjusted for ﬂuid intake and urine volume, and
used to calculate sweat loss, which was then normalized to
body surface area and presented as the hourly sweat rate
(SR). Relative perceived exertion (RPE) was assessed every
15 min during the HTT using the Borg scale (Borg, 1998),
and a scale from 1 to 13 (unbearably cold to unbearably
hot sensation, respectively), was used to rate the subjective
sensation of thermal comfort (Thermal comfort rate, TCR).
Safety thresholds for test cessation were set at Trec = 39◦C or
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Astaxanthin Exercise Recovery and Heat Tolerance
HR = 180 bpm, at the study physician’s discretion or at the
participant’s request.
Maximal oxygen uptake (VO2 Max) was determined by cardio
pulmonary exercise testing (CPET), using a modiﬁed Bruce
protocol composed of 5 min seated rest, followed by 5 min of
walking on a treadmill at 5 kph, and 2% incline, followed by
running at 9 kph, with an incrementally increasing incline (2%
every 2 min), until reaching VO2 Max, which was determined by
3 of 4 criteria during the test: (1) leveling oﬀ of the VO2 curve
to a plateau, (2) reaching >90% of the participant’s predicted
maximal heart rate (210–0.65 × Age), (3) reaching a respiratory
exchange ratio (RER) ≥1.1 or 4) at the participant’s request,
after reaching a subjective state of extreme physical tiredness.
Additional supportive indications after test completion were
reaching >8 mmol/l of blood lactate, or RPE > 17 (Edvardsen
et al., 2014; Debeaumont et al., 2016). The ventilatory anaerobic
threshold (AT) was determined visually by two trained examiners
according to the American heart association guidelines (Balady
et al., 2010). Continuous monitoring lasted throughout recovery,
which consisted of 3 min at 5 kph and 2% incline, followed by
3 kph at 0% incline, and ﬁnally, 1 min, seated. The test was
performed on a CPET machine (ZAN 600, Nspire Health, USA)
connected to a treadmill ergometer (Model 770 S, RAM medical
and industrial instruments, Germany). Reaching onset of blood
lactate accumulation (OBLA) was conﬁrmed by examining blood
lactate level before and after the test (lactate scout+ analyzer,
Sports Resource Group Inc., USA). Heart rate was continuously
monitored by a heart rate monitor (model: RS800CX, POLAR,
Finland). Assessment of RPE took place before and after the VO2
Max test.
Blood was drawn on physical testing days before the VO2 Max
test and on HTT days before, immediately after and at 60 min
after the end of the HTT. Blood was collected in yellow gel
chemistry collection tubes (Becton, Dickinson and Co., NJ, USA),
allowed to clot for 30 min and centrifuged. Serum was separated
immediately and stored at −80◦C pending analysis. Serum lipid
and triglyceride (TG) proﬁle, CRP, and CPK were analyzed by the
central laboratories at the Sheba medical center. A commercially
available ELISA kit for High-Sensitivity HSP72 detection was
used to measure serum HSP72 levels in optical density (OD),
which was used to calculate the HSP72 concentration in ng/ml,
according to the manufacturer’s instructions (AMP’D R⃝ HSP70
high sensitivity ELISA kit, ENZ-KIT-101, Enzo life sciences,
NY, USA).
STATISTICS
Anthropometric, physiological and biochemical parameters were
statistically analyzed using the SPSS software (version 23, IBM,
USA). Treatments and time point were taken as the independent
variable and participants were considered a random sample of the
general population. Normality of distribution was assessed by the
Kolomogorov-Smirnov test and comparison between treatment
groups and between pre- and post-supplementation time points
was made with 1-way ANOVA, with Tukey post hoc analysis
for normally distributing variables, or Mann-Whitney U-test for
non-normally distributing variables. Analysis of the diﬀerence
in the change in parameters due to supplementation between
treatment groups was conducted by calculating the delta between
the pre- and post-supplementation states (pre-supplemented
state subtracted from the post-supplemented state). Normality
of distribution was assessed by the Kolomogorov-Smirnov test
and comparison between treatment groups was made by T-test
for normally distributing variables and Mann-Whitney U-test
for non-normally distributing variables. Leven’s test was used
to evaluate the equality of variance between treatment groups,
followed by the appropriate Student’s t-test (2-tailed) to assess
signiﬁcance. In order to assess the signiﬁcance of diﬀerence
between repetitive blood tests, ANOVA for repeated measures
followed by Bonferroni post-hoc analysis or Friedman’s omnibus
test followed by Wilcoxon’s signed-rank test with Bonferroni
adjustment were used, for normally or non-normally distributing
variables, respectively. A signiﬁcant p-value was set at 0.05.
RESULTS
Table 1 lists key parameters of aerobic capacity, as recorded by
the VO2 Max test. In both groups, anaerobic threshold (AT)
was achieved at approximately 72% of the VO2 Max value, VO2
Max was similar and did not improve post-supplementation.
Aerobic characteristics did not diﬀer between the ATX and PLA
groups both before and after supplementation, as seen in the
unchanged AT, maximal oxygen uptake, reduction in heart rate
during recovery, and in substrate utilization demonstrated by the
scatter plot of respiratory exchange ratio (RER) vs. oxygen uptake
(VO2) (Supplemental Figure 1).
However, a signiﬁcant diﬀerence was observed between
the two groups post supplementation in the blood lactate
concentration measured after the VO2 Max test. Additionally,
a signiﬁcant reduction was observed in oxygen uptake at the
end of recovery between the pre-supplementation and post-
supplementation time points in the ATX group compared to the
PLA group (Table 1). Supplemental Figure 2 depicts the VO2
values during the test by group, before and after supplementation.
Table 2 lists the results from the HTT. The physiological
parameters monitored continuously during the test, including
HR, Trec, and Tsk displayed no signiﬁcant diﬀerence between
the ATX and PLA groups. During the ﬁrst, un-supplemented
HTT, and the second, supplemented HTT, Basal Trec in both
groups was below 37◦C, and increased by about 1◦C. Heart rate
began at nearly 80 bpm and increased to just under 120 bpm
in both groups.
Pre-supplementation sweat rate in the PLA group was
signiﬁcantly higher than the ATX group (which disappeared
post-supplementation), and post-supplementation in the PLA
group (p < 0.001). The subjective scales representing sensations
of relative perceived exertion (RPE) and thermal comfort (TCR),
which were monitored every 15 min during the test and for 1 h
after its completion, also displayed no diﬀerence between groups
or exposures. Participants perceived a mild to moderate eﬀort in
reporting their subjective sensations in the Borg scale (RPE) and
moderate heat in the TCR scale.
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Astaxanthin Exercise Recovery and Heat Tolerance
TABLE 1 | Main VO2 Max ﬁndings: This table lists the main ﬁndings from the maximal oxygen uptake tests performed before (pre) and after (post) supplementation in the two study groups.
Test parameter
ATX pre
ATX post
PLA pre
PLA post
ANOVA/Mann-Whitney U
Delta ATX
Delta PLA
T-test/
Mann-Whitney U
Mean ± St. Error
Mean ± St. Error
Mean ± St. Error
Mean ± St. Error
p-value
Post hoc
Tukey p-value
Mean ± St. Error
Mean ± St. Error
p-value
RPE Before
6 ± 0
7 ± 0
7 ± 1
7 ± 0
N.S
0 ± 1
−2 ± 1
N.S
RPE After
18 ± 1
17 ± 0
16 ± 1
17 ± 0
N.S
−2 ± 2
−3 ± 3
N.S
1 RPE
12 ± 1
11 ± 0
9 ± 1
11 ± 1
N.S
−2 ± 1
−1 ± 2
N.S
BLA before (mmole.l−1)
2.3 ± 0.31
2.57 ± 0.21
2.04 ± 0.24
2.08 ± 0.2
N.S
0.27 ± 0.37
−0.17 ± 0.38
N.S
BLA after (mmole.l−1)
13.46 ± 0.94
11.92 ± 0.85
12.65 ± 0.68
15.09 ± 1.02
N.S
−1.54 ± 0.85
0.93 ± 1.84
N.S
1 BLA (mmole.l−1)
11.16 ± 0.78
9.35 ± 0.89
10.61 ± 0.73
13.01 ± 1.05
0.044
0.027*
−1.81 ± 0.89
1.1 ± 1.67
N.S
Sys. BP before (mmHg)
119 ± 2.77
120.64 ± 3.96
118.5 ± 2.87
121.29 ± 2.9
N.S
−8.42 ± 10.34
−33.6 ± 19.81
N.S
Dias. BP before (mmHg)
78.58 ± 3.14
77.64 ± 3.65
72.7 ± 3.09
79.57 ± 5.07
N.S
−7.42 ± 8.94
−17 ± 14.49
N.S
Sys. BP after (mmHg)
118.9 ± 4.42
127.2 ± 3.84
122.9 ± 5.34
117.71 ± 5.51
N.S
6.92 ± 21.73
−40.5 ± 17.89
N.S
Dias. BP after (mmHg)
75.1 ± 2.93
79.4 ± 3.11
76 ± 3.24
73.29 ± 4.36
N.S
3.58 ± 14.96
−24.7 ± 11.12
N.S
AT VO2 (ml.kg−1.min−1)
37.51 ± 3.35
40.1 ± 1.97
37.27 ± 3.51
38.53 ± 3.98
N.S
2.59 ± 3.68
1.26 ± 2.09
N.S
AT (% of VO2 Max)
72.32 ± 6.08
78.27 ± 3.17
71.3 ± 3.77
73.23 ± 4.51
N.S
5.95 ± 6.86
1.94 ± 3.79
N.S
Max load (Watt)
284.67 ± 11.46
288.5 ± 12.66
276.7 ± 19.6
281.1 ± 19.06
N.S
3.83 ± 5.92
4.4 ± 2.61
N.S
VO2 Max (ml.kg−1.min−1)
52.55 ± 1.83
51.24 ± 1.6
51.49 ± 2.32
51.54 ± 2.66
N.S
−1.31 ± 0.7
0.05 ± 0.71
N.S
HR Max (bpm)
190.67 ± 2.85
191.17 ± 2.33
191.2 ± 2.62
190.1 ± 2.85
N.S
0.5 ± 1.58
−1.1 ± 1.42
N.S
RER Max
1.14 ± 0.02
1.13 ± 0.02
1.15 ± 0.02
1.15 ± 0.03
N.S
0 ± 0.02
0 ± 0.02
N.S
#End recov. VO2 (ml*kg−1*min−1)
12.08 ± 0.32
10.76 ± 0.45
11.43 ± 0.43
11.85 ± 0.51
N.S
−2.22 ± 0.99
0.42 ± 0.48
##0.006
#End recov. VO2 (% of VO2 Max)
23.52 ± 0.64
21.31 ± 0.68
22.44 ± 0.98
23.27 ± 1.04
N.S
−2.02 ± 0.64
0.83 ± 0.79
0.01
1#end recov. HR (bpm)
47.25 ± 2
43.08 ± 2.24
40.3 ± 4
40.3 ± 4.7
N.S
−4.17 ± 2.15
0 ± 5.19
N.S
RPE, relative perceived exertion; BLA, blood lactate; Sys., systolic; Dias., diastolic; BP, blood pressure; AT, Anaerobic threshold; Max, maximal; HR, heart rate; bpm, beats per minute; recov., recovery.
#End recovery is the average value of the last 30 s recorded while seated at the end of the test.
*Between ATX post and PLA post.
##Value calculated with Mann-Whitney U-test, for non-normally distributing variables.
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TABLE 2 | Main HTT ﬁndings: This table lists the main ﬁndings from the HTT performed before (pre) and after (post) supplementation in both treatment groups.
Test parameter
ATX pre
ATX post
PLA pre
PLA post
ANOVA/Mann-Whitney U
Delta ATX
Delta PLA
T-test/
Mann-Whitney U
Mean ± St. Error
Mean ± St. Error
Mean ± St. Error
Mean ± St. Error
p-value
post hoc Tukey p-value
Mean ± St. Error
Mean ± St. Error
p-value
Basal Trec (◦C)
36.93 ± 0.08
36.87 ± 0.08
36.95 ± 0.07
36.82 ± 0.12
N.S
−3.13 ± 3.04
−0.12 ± 0.12
N.S
Max Trec (◦C)
37.79 ± 0.1
37.9 ± 0.1
37.79 ± 0.1
37.91 ± 0.17
N.S
−3.05 ± 3.12
0.12 ± 0.16
N.S
1 Trec (◦C)
0.83 ± 0.09
0.93 ± 0.12
0.9 ± 0.1
1.1 ± 0.18
N.S
0.1 ± 0.09
0.2 ± 0.2
N.S
End + 1 h Trec (◦C)
37.29 ± 0.06
37.26 ± 0.13
37.26 ± 0.07
37.19 ± 0.14
N.S
3.07 ± 3.13
7.38 ± 4.97
N.S
Basal Tskin (◦C)
35.41 ± 0.21
35.12 ± 0.24
35.68 ± 0.24
35.49 ± 0.24
N.S
−0.26 ± 0.2
−7.29 ± 4.76
N.S
Max Tskin (◦C)
36.4 ± 0.14
36.43 ± 0.23
36.12 ± 0.17
36.28 ± 0.29
N.S
−3 ± 3.01
−7.1 ± 4.83
N.S
1 Tskin (◦C)
0.99 ± 0.22
−2 ± 3.41
0.4 ± 0.27
0.67 ± 0.37
N.S
−2.74 ± 3.07
0.2 ± 0.36
N.S
Sweat rate*BSA−1
−342.81 ± 23.25
−334.17 ± 22.53
−472.89 ± 29.72
−333 ± 19.71
<0.001
0.002*
8.64 ± 36.11
139.89 ± 39.16
0.023
HR Start (bpm)
79.17 ± 3.29
79.67 ± 3.17
80.4 ± 4.37
79.1 ± 3.84
N.S
0.5 ± 2.91
−1.3 ± 2.78
N.S
HR end (bpm)
118 ± 3.84
116.08 ± 4.71
117.7 ± 6.38
118.9 ± 5.47
N.S
−1.92 ± 4.15
1.2 ± 3.57
N.S
1 HR (bpm)
38.83 ± 2.29
36.42 ± 5.09
37.3 ± 5.11
39.8 ± 3.68
N.S
−2.42 ± 5.32
2.5 ± 3.42
N.S
RPE start
8 ± 1
7 ± 0
7 ± 0
7 ± 0
N.S
0 ± 1
0 ± 1
N.S
RPE end
11 ± 1
8 ± 0
9 ± 1
10 ± 2
N.S
−1 ±
0 ± 2
N.S
TCR start
9 ± 1
7 ± 1
8 ± 0
8 ± 1
N.S
−1 ± 1
1 ± 1
N.S
TCR end
11 ± 1
11 ± 1
9 ± 1
9 ± 0
N.S
0 ±
0 ± 1
N.S
MAP Start (mmHg)
83.78 ± 2.72
89.39 ± 5.03
80.57 ± 9.23
83.73 ± 2.36
N.S
5.61 ± 5.19
3.17 ± 8.96
N.S
MAP end (mmHg)
81.33 ± 2.36
80.36 ± 2.96
83.8 ± 3.69
80.97 ± 2.01
N.S
1.27 ± 2.92
−2.83 ± 4.7
N.S
1 MAP (mmHg)
−9.22 ± 6.94
−9.03 ± 5.13
3.23 ± 11.59
−2.77 ± 2.4
N.S
0.19 ± 7.92
−6 ± 10.89
N.S
MAP HTT + 1 h (mmHg)
83.48 ± 2.47
90.42 ± 1.23
87.6 ± 4.01
85.17 ± 2.24
N.S
6.67 ± 2.41
−2.43 ± 4.46
N.S
Max, maximal; Trec, rectal temp.; Tskin, skin temp.; BSA, body surface area; HR, heart rate; bpm, beats per minute; RPE, relative perceived exertion; TCR, thermal comfort rate; MAP, Mean arterial pressure. *Between PLA pre and ATX
pre and between PLA pre and PLA post.
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Astaxanthin Exercise Recovery and Heat Tolerance
Biochemical analyses:
Table 3 depicts measured
serum
concentrations of CRP, CPK, HSP72, and the lipid proﬁle,
including,
high
density
lipoproteins
(HDL),
low
density
lipoproteins
LDL
total
cholesterol
and
Triglycerides.
No
signiﬁcant diﬀerences were observed between the ATX and
PLA groups in the serum levels of HSP72 protein, in the lipid
and triglyceride proﬁle, in CRP or in CPK concentrations, both
before and after the eﬀort. However, during all HTT testing days,
CPK levels obtained before the test were signiﬁcantly lower than
those obtained immediately after the test, in both groups, both
before and after supplementation.
DISCUSSION
We examined the inﬂuence of 1 month of 12 mg daily
Astaxanthin supplementation on heat tolerance and aerobic
capacity. Astaxanthin improved exercise recovery but had no
inﬂuence on performance in the heat.
Human exercise models, in contrast to animal studies have
shown conﬂicting results regarding the eﬀects of Astaxanthin on
performance. For example: the beneﬁcial eﬀects of Astaxanthin in
competitive cyclists shown while consuming 4 mg/day (Earnest
et al., 2011), vs. no signiﬁcant diﬀerence in performance of
well-trained cyclists while consuming 20 mg/day (Res et al.,
2013). Neither metabolic markers nor blood biochemistry of
human cohorts revealed dose or time dependent metabolic
changes attributable to Astaxanthin supplementation (Karppi
et al., 2007; Earnest et al., 2011; Res et al., 2013). The variance of
substrate oxidation proﬁles during exercise existing in the general
population and the steady state nature of the measurement
may have masked a metabolic supplementation eﬀect. The
graded VO2 Max test used in our study, designed to answer
the questions raised regarding the inﬂuence of Astaxanthin on
substrate utilization in exercising humans over a range of exercise
intensities (Brown et al., 2018), showed no eﬀect on aerobic
capacity or its components: energy substrate use during the VO2
Max test displayed no supplementation eﬀect to inﬂuence fat
utilization over carbohydrates in either group, as demonstrated
in Supplemental Figure 1.
However, the change in blood lactate concentration after the
VO2 Max test (Table 1), along with the signiﬁcant reduction
in oxygen uptake at the end of recovery in the ATX group
compared to the PLA group, may suggest less oxidative stress
and faster recovery in comparison with the control, which
is a possible advantage for Astaxanthin supplementation. In
Supplemental Figure 2, a more rapid return to lower VO2 values
during recovery is seen in ATX after supplementation compared
to before supplementation.
Though evidence from animal models suggests that post
exercise recovery may improve with Astaxanthin administration,
particularly, by diminishing exercise induced tissue damage
markers such as creatine kinase (CK) and myeloperoxidase
(MPO), through anti-oxidative and anti-inﬂammatory pathways
(Aoi et al., 2003; Guo et al., 2018), human studies are ambiguous:
muscle soreness, exercise force production and plasma CK
displayed no signiﬁcant diﬀerence in highly trained individuals
who received 3 weeks of 4 mg/day Astaxanthin (Bloomer
et al., 2005). However, longer supplementation (90 days) in
young soccer players was associated with improved indirect
damage markers like reduced lactate dehydrogenase (LDH),
and non-signiﬁcant improvements in CK and inﬂammatory
markers including CRP and leukocyte and neutrophil counts
(Djordjevic et al., 2012). Validated information on the eﬀects
of Astaxanthin supplementation on exercise performance and
recovery, particularly in diverse populations, is lacking.
In the present experiment, though exercise recovery of
oxygen uptake was improved in the Astaxanthin group post-
supplementation, contrastingly, serum inﬂammation (CRP),
muscle damage (CPK) and lipid proﬁle remained unaﬀected by
supplementation in both groups.
The physiological strain induced by the HTT, was mild for
both groups (Trec < 38◦C and HR < 120 bpm), as supported
by the lack of change in HSP72 post-exercise, pointing to
an insuﬃcient perturbation of the thermoregulatory system
and the absence of an HSR. Notably, experimental conditions,
particularly the physiological safety thresholds, were limited
by ethical constraints, and could not induce a higher thermal
threshold. Under the experimental conditions employed in
this study, no participant reached the safety threshold during
heat exposure.
An Additional component contributing to the observed
physiological response may have been the ﬁtness level of
participants and the relatively mild eﬀort undertaken by them
during the HTT. An average VO2 Max of 51–52 ml × kg−1 ×
min−1 was typical of the study participants. The average HR
elevation during the HTT was ∼40 bpm, reﬂecting a 21% change
relative to the measured maximal HR in the VO2 Max test
(Table 1), indicating a state of mild stress experienced during the
HTT across treatment groups and exposures.
Nevertheless, the signiﬁcant diﬀerence in CPK levels from the
beginning to the end of the HTT, in both groups indicates some
muscle damage resulting from the HTT, which was unaﬀected by
supplementation (Table 3).
The signiﬁcantly higher sweat rate in the pre-supplemented
PLA group compared to PLA post-supplementation and to ATX
pre- and post-supplementation cannot be explained by an eﬀect
of supplementation, and can only be attributed to a diﬀerence
between participant groups. This was, however, insigniﬁcant
when the change in sweat rate from pre- to post-supplementation
was compared between treatment groups (Table 2).
The daily dose of Astaxanthin used in this work (12 mg) was
reﬂective of the highest recommended dose for humans at the
time, which has been substantially increased since then to 24 mg
daily (Visioli and Artaria, 2017). Consumption of a larger dose
may have evoked greater eﬀects in aerobic function and cellular
protective aspects important to coping with the damages of heat
stress exposure.
CONCLUSION
Preemptive nutritional supplementation is a promising avenue
for exercise science research as a way of improving physiological
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Astaxanthin Exercise Recovery and Heat Tolerance
TABLE 3 | Serum levels of CPK (mg/Liter), CRP (mg/Liter), HSP72 (ng/mL), and lipid proﬁle: HDl, LDL, total cholesterol and triglycerides (mg/dL).
Supp.
Time point
ATX
PLA
T-test\Mann-Whitney U
Mean ± St. Error
Mean ± St. Error
CRP pre-supp.
Before HTT
1.09 ± 0.32
0.6 ± 0.14
N.S
After HTT
1.08 ± 0.33
0.63 ± 0.14
After HTT + 1 h
1.06 ± 0.3
0.62 ± 0.13
#1 HTT
−0.01 ± 0.03
0.03 ± 0.01
##1 HTT + 1 h
−0.03 ± 0.03
0.02 ± 0.02
CRP post-supp.
Before HTT
1.54 ± 0.46
1.31 ± 0.79
After HTT
1.53 ± 0.45
1.22 ± 0.72
After HTT + 1 h
1.55 ± 0.46
1.25 ± 0.76
#1 HTT
−0.01 ± 0.04
−0.08 ± 0.07
##1 HTT + 1 h
0.01 ± 0.07
−0.05 ± 0.03
CPK pre-supp.
Before HTT
221.42 ± 61.68
188.7 ± 36.69
After HTT
260.09 ± 69.54
208 ± 39.68
After HTT + 1 h
234.42 ± 57.17
198.8 ± 33.96
#1 test
27.64 ± 9.4
19.3 ± 5.06
##1 recovery
−15 ± 8.21
−9.2 ± 4.64
CPK post-supp.
Before HTT
191.5 ± 42.54
134.5 ± 25.83
After HTT
207.42 ± 43.94
151.3 ± 26.3
After HTT + 1 h
204.75 ± 43.39
146.1 ± 25.9
#1 test
15.08 ± 4.7
16.8 ± 3.11
##1 recovery
−2.17 ± 3.35
−5.2 ± 2
HSP72 pre-supp.
Before HTT
1.9 ± 0.75
4.06 ± 0.62
0.021
After HTT
2.68 ± 0.88
3.77 ± 0.67
N.S
**1 HTT
0.77 ± 0.54
−0.3 ± 0.53
HSP72 post-supp.
Before HTT
2.25 ± 0.79
3.75 ± 0.77
After HTT
2.31 ± 0.76
3.77 ± 0.77
**1 HTT
0.06 ± 0.09
0.03 ± 0.09
HSP72 *1Supp.
1 before HTT
0.35 ± 0.12
−0.32 ± 0.34
1 after HTT
−0.37 ± 0.59
0.01 ± 0.78
Total cholesterol
Pre-supp.
139 ± 9.82
156.6 ± 5.98
Post-supp.
144.5 ± 7.12
160.5 ± 7.12
*1
5.5 ± 8.14
3.9 ± 5.12
Triglycerides
Pre-supp.
88.25 ± 16.18
98 ± 11.62
Post-supp.
94.33 ± 15.78
95.2 ± 11.62
*1
6.08 ± 8.25
−2.8 ± 9.88
HDL
Pre-supp.
46 ± 2.7
47.2 ± 2.92
Post-supp.
46.42 ± 2.04
47.8 ± 2.12
*1
0.42 ± 1.66
0.6 ± 1.42
LDL
Pre-supp.
96.17 ± 5.29
105.7 ± 6.29
Post-supp.
94.42 ± 5.68
108.8 ± 6.69
*1
−1.75 ± 3.85
3.1 ± 3.74
n = 12 in the ATX group, and 10 in the PLA group.
HSP72 protein levels were measured in eight participants of each group using ELISA before the HTT and 1 h after completion of the test. Analysis was made in triplicate and averaged
for each measurement.
#Calculated by subtracting before HTT from after HTT values.
##Calculated by subtracting before HTT from after HTT + 1 h values.
*Supplementation delta was calculated by subtracting pre supplementation values from the post supplementation values for the appropriate time point.
**Delta (1) at each HTT was calculated by subtracting Before HTT results from the After HTT results.
Supp., supplementation; HTT, heat tolerance test; 1, delta, HDL, high density lipoproteins; LDL, low density lipoproteins.
For CRP, Friedman’s omnibus test between time points within treatments revealed no statistically signiﬁcant difference.
For CPK, 1-way ANOVA revealed no signiﬁcant difference for any of the time points between the ATX and PLA groups. Friedman’s omnibus test, followed by Wilcoxon signed-ranks
test revealed signiﬁcant differences between CPK values before and after the HTT (p = 0.005 and p < 0.01 in the ATX and PLA groups, respectively) and in the ATX group, between
after the HTT and 1 h after its completion (p = 0.006).
Analysis of HSP72 with Wilcoxon signed ranks test revealed no signiﬁcant difference between measurements within treatment groups.
Analysis of TG’s by Wilcoxon signed-ranks test revealed no signiﬁcant difference between time points within treatment groups.
Analysis of Cholesterol and lipids by paired samples t-test revealed no signiﬁcant difference between measurements within treatment groups.
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Fleischmann et al.
Astaxanthin Exercise Recovery and Heat Tolerance
resilience in preparation for an anticipated exposure to
adverse
conditions
and
to
strenuous
eﬀorts.
Long
term
supplementation of 12 mg\daily Astaxanthin contributed to
improved aerobic recovery, but was not beneﬁcially manifested
under the examined heat load conditions. It remains to
be seen if administration of larger doses of Astaxanthin or
exposure to greater environmental and physiological stress
that elicit a heat shock response might bring additional
protective
mechanisms
of
Astaxanthin
supplementation
into light.
DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualiﬁed researcher.
AUTHOR CONTRIBUTIONS
CF contributed to the conception and design of the study,
conducted the experiments, analyzed the data, and wrote
the manuscript. MH contributed to data analysis and to
manuscript design and reviewed the manuscript. RY contributed
to conducting the experiments, to data analysis, and reviewed
the manuscript. HR participated as the study nutritionist and
contributed to conducting the experiments, to data analysis,
and reviewed the manuscript. YH contributed to the conception
and design of the study, to conducting the experiments, and
reviewed the manuscript.
FUNDING
This work was funded by the IDF medical corps research fund,
grant number: 44405899192.
ACKNOWLEDGMENTS
The authors would like to acknowledge the participants who took
part in this study.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fspor.
2019.00017/full#supplementary-material
Supplemental Figure S1 | Scatter plot of RER vs. VO2 during the VO2 Max
tests, for the ATX and PLA supplementation groups. The ATX group before and
after supplementation is represented by the blue and gray dots, respectively. The
PLA group before and after supplementation is represented by the orange and
yellow dots, respectively.
Supplemental Figure S2 | VO2 Max test. Depicts the VO2 Max test graphs by
group and stage: (A) upper left, green lines: ATX group, before supplementation;
(B) lower left, brown lines: ATX group, after supplementation; (C) upper right, blue
lines: PLA group, before supplementation; (D) lower right, black lines: PLA group,
after supplementation.
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Conﬂict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or ﬁnancial relationships that could
be construed as a potential conﬂict of interest.
Copyright © 2019 Fleischmann, Horowitz, Yanovich, Raz and Heled. This is an
open-access article distributed under the terms of the Creative Commons Attribution
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Frontiers in Sports and Active Living | www.frontiersin.org
11
September 2019 | Volume 1 | Article 17

Asthaxanthin Improves Aerobic Exercise Recovery Without Affecting Heat Tolerance in Humans.
Fleischmann, Chen,Horowitz, Michal,Yanovich, Ran,Raz, Hany,Heled, Yuval