Physiological Reports. 2021;9:e14760. 
 
  | 1 of 11
https://doi.org/10.14814/phy2.14760
wileyonlinelibrary.com/journal/phy2
Received: 18 November 2020 | Revised: 22 January 2021 | Accepted: 23 January 2021
DOI: 10.14814/phy2.14760  
O R I G I N A L  A R T I C L E
Four weeks of high- intensity training in moderate, but not mild 
hypoxia improves performance and running economy more 
than normoxic training in horses
Kazutaka Mukai1
 |   Hajime Ohmura1 |   Yuji Takahashi1
 |   Yu Kitaoka2
 |   
Toshiyuki Takahashi1
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original 
work is properly cited.
© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society
These data were presented in abstract form and as a poster presentation at the American College of Sports Medicine Conference on Integrative Physiology of 
Exercise, San Diego, USA, September 2018. 
1Equine Research Institute, Japan Racing 
Association, Shimotsuke, Japan
2Kanagawa University, Yokohama, 
Kanagawa, Japan
Correspondence
Kazutaka Mukai, Equine Research 
Institute, Japan Racing Association, 
1400- 4 Shiba, Shimotsuke, Tochigi 329- 
0412, Japan.
Email: mukai@equinst.go.jp
Funding information
This study was funded by the Japan 
Racing Association.
Abstract
We investigated whether horses trained in moderate and mild hypoxia demonstrate 
greater improvement in performance and aerobic capacity compared to horses trained 
in normoxia and whether the acquired training effects are maintained after 2 weeks of 
post- hypoxic training in normoxia. Seven untrained Thoroughbred horses completed 
4 weeks (3 sessions/week) of three training protocols, consisting of 2- min cantering 
at 95% maximal oxygen consumption 
( ̇VO2max
)
 under two hypoxic conditions (H16, 
FIO2 = 16%; H18, FIO2 = 18%) and in normoxia (N21, FIO2 = 21%), followed by 
2 weeks of post- hypoxic training in normoxia, using a randomized crossover study 
design with a 3- month washout period. Incremental treadmill tests (IET) were con-
ducted at week 0, 4, and 6. The effects of time and groups were analyzed using mixed 
models. Run time at IET increased in H16 and H18 compared to N21, while speed 
at ̇VO2max was increased significantly only in H16. ̇VO2max in all groups and cardiac 
output at exhaustion in H16 and H18 increased after 4 weeks of training, but were 
not significantly different between the three groups. In all groups, run time, ̇VO2max, 
V ̇VO2max, ̇Qmax, and lactate threshold did not decrease after 2 weeks of post- hypoxic 
training in normoxia. These results suggest that 4  weeks of training in moderate 
(H16), but not mild (H18) hypoxia elicits greater improvements in performance and 
running economy than normoxic training and that these effects are maintained for 
2 weeks of post- hypoxic training in normoxia.
K E Y W O R D S
horse, hypoxic training, performance, running economy
1 | INTRODUCTION
Altitude/hypoxic training is popular in endurance athletes 
and has been used recently in middle- distance runners, 
swimmers, and speed skaters. Although the efficacy of al-
titude/hypoxic training for sea- level exercise performance 
remains controversial from a research perspective, athletes 
continue to use it to train for competitions. Most commonly, 
2 of 11 |   
MUKAI et Al.
athletes both live and train at moderate to high altitude 
(live high- train high, LHTH) or live at altitude and train 
at sea level (live high- train low, LHTL). Previous reports 
and reviews have shown increases in exercise performance, 
maximal oxygen consumption 
( ̇VO2max
)
, and hemoglobin 
mass after several weeks of LHTH and/or LHTL training 
(Bonetti & Hopkins, 2009; Millet et al., 2010; Robertson 
et al., 2010).
Another hypoxic training program gaining popularity is 
the live low- train high (LLTH) model. This model involves 
athletes living in normoxia and performing some training 
sessions in hypoxia. While several LLTH studies failed to 
demonstrate benefits in LLTH compared with equivalent 
normoxic training (McLean et al., 2014), some studies 
demonstrated that LLTH training can enhance exercise per-
formance, maximal workload, and 
( ̇VO2max
)
 (Czuba et al., 
2011), and can augment skeletal muscle mitochondrial 
density, capillary- to- fiber ratio, and fiber cross- sectional 
area (Desplanches et al., 1993; Vogt et al., 2001), likely 
via up- regulation of hypoxia- inducible factor 1α (HIF- 1α)
(Vogt et al., 2001). Some authors also suggest that LLTH 
may improve anaerobic exercise performance (Hamlin 
et al., 2010; Hendriksen & Meeuwsen, 2003), possibly via 
increases in muscle buffering capacity (Gore et al., 2001) 
and increased glycolytic enzyme activity (Puype et al., 
2013).
When athletes and coaches use hypoxic training in prac-
tical situations, a key question is when is the best timing to 
return to sea level before a race to optimize performance. The 
general consensus among top coaches suggests that endur-
ance performance is optimized after 14 days at sea level after 
altitude/hypoxic training (Dick, 1992), but there is limited 
scientific evidence to support this opinion. While some re-
searchers suggest that repeated sprint ability and hemoglobin 
mass are higher at 3 weeks after hypoxic training compared 
with pre- hypoxic training levels (Brocherie et al., 2015), an-
other group reported that most hematological adaptations 
after altitude training are lost in 9 days (Pottgiesser et al., 
2012). In addition, previous studies on the maintenance of 
post- hypoxic training use mostly LHTL training and not 
LLTH.
Thoroughbred horses have high ̇VO2max, exceeding 
180  mL/kg/min in trained individuals, and the aerobic 
contribution to total energy expenditure for a 120- s sprint 
is estimated to reach >70% (Eaton et al., 1995; Ohmura 
et al., 2010). Furthermore, Thoroughbred horses have 
large amounts of glycogen (>600 mmol/kg dwt) in their 
muscle (Davie et al., 1999) and the lactate concentration 
in plasma and skeletal muscle during maximal exercise 
increases to more than 20 mmol/L and 20 mmol/kg, re-
spectively (Kitaoka et al., 2014), which suggests that 
Thoroughbred horses also utilize the glycolytic pathway 
maximally for energy resources during high- intensity 
exercise. Therefore, improvements in both aerobic and 
anaerobic capacity are needed to enhance equine racing 
performance. Previously, we reported that high- intensity 
training (100% ̇VO2max for 3  min, 3 sessions/week for 
4  weeks) in moderate hypoxia (15% O2) improves run 
time and ̇VO2max at incremental exercise tests (IET) in 
 normoxia to a greater extent than the same training in nor-
moxia (Mukai et al., 2020), which  indicates that  hypoxic 
training may be a strong strategy for better exercise 
 performance without increasing absolute training speed 
and/or distance. However, very few  studies have examined 
hypoxic training in horses (Davie et al., 2017; Ohmura 
et al., 2017), and further studies are needed to determine 
the optimal severity of hypoxia and the intensity, duration, 
and volume of training in hypoxia.
The purpose of this study was to investigate the hypoth-
esis that horses trained in moderate and mild hypoxia for 
4  weeks experience greater improvements in performance 
and aerobic capacity compared with horses trained in nor-
moxia. In addition, we examined whether acquired training 
effects are maintained after 2 weeks of post- hypoxic training 
in normoxia.
2 | MATERIALS AND METHODS
Protocols for the study were reviewed and approved by the 
Animal Welfare and Ethics Committee of the Japan Racing 
Association (JRA) Equine Research Institute (Permit number: 
2017– 1, 2018– 1). All surgery was performed under sevoflu-
rane anesthesia and all incisions for catheter placements were 
performed under local anesthesia using lidocaine. All efforts 
were made to minimize animal suffering.
2.1 | Horses
Seven untrained Thoroughbreds (2 geldings and 5 females; 
mean ±SE age, 7.9 ± 0.7 years; body weight, 512 ± 11 kg 
at the onset of the study) were used in this study. Each 
horse had a carotid artery moved surgically from the ca-
rotid sheath to a subcutaneous location under sevoflu-
rane anesthesia to facilitate arterial catheterization. After 
 recovery from surgery, the horses were trained to run on a 
treadmill (Sato I, Sato AB, Uppsala, Sweden) while wear-
ing an open- flow mask (Pascoe et al., 1999). After surgery, 
each horse was kept in a 17 x 22 m yard for approximately 
6 h/day every day for at least 4 months before treadmill 
experiments began. All horses received 1 kg of oats, 1 kg 
of pelleted feed, and 3 kg of timothy hay in the morning, 
and 1 kg of oats, 2 kg of pelleted feed, and 3 kg of timothy 
hay in the afternoon. Water was available ad libitum during 
the study.
   | 3 of 11
MUKAI et Al.
2.2 | Experimental design
In a randomized crossover design, horses were trained in 
moderate hypoxia (H16; 16% inspired O2), mild hypoxia 
(H18; 18% inspired O2), or normoxia (N21; 21% inspired 
O2) for 3 days/week on a treadmill at a 6% incline. The 
horses were pastured in 17 × 22 m yards for approximately 
6 h/day and walked for 1 h/day in a walker on the other 
4 days during the training period. The training session con-
sisted of a warm- up (walking at 1.7 m/s for 30 min and 
trotting at 4 m/s for 2 min), cantering at 7 m/s for 1 min 
and for 2 min at the speed previously determined to elicit 
95% ̇VO2max measured in normoxia, followed by a cool- 
down (1.7 m/s for 30 min) in all groups. In hypoxic groups, 
horses wore an open- flow mask after walking for 30 min 
and were exposed to hypoxia during trotting for 2 min and 
cantering at 7 m/s for 1 min and at 95% ̇VO2max for 2 min. 
After 4  weeks of hypoxic/normoxic training, all groups 
continued the same training protocols in normoxia for 
2 weeks. Each training period was separated by 3 months 
to ensure a sufficient detraining interval.
2.3 | Incremental exercise tests (IET) 
in normoxia
Incremental exercise tests in normoxia were conducted at 
weeks 0, 4, and 6. The procedure for the incremental exercise 
test, including oxygen consumption measurements and blood 
sampling, has been described previously (Mukai et al., 2017). 
Briefly, after catheters and transducers were connected and 
tested, the horse began its exercise. The horse warmed up 
by trotting at 4 m/s for 3 min, then cantering up a 6% incline 
for 2 min each at 1.7, 4, 6, 8, 10, 12, 13, and 14 m/s until 
the horse could not maintain its position at the front of the 
treadmill with humane encouragement. This condition was 
defined as exhaustion. Run time to exhaustion was meas-
ured with a stopwatch. For each speed, the horse ran on the 
treadmill for 90 s to allow the O2 transport system to come 
to steady- state (equine ̇VO2 comes to steady- state faster than 
human ̇VO2 does), then ̇VO2 was calculated for the final 30 s 
of each step. Heart rate was recorded using a commercial 
heart rate monitor (S810, Polar, Kempele, Finland) and mean 
heart rate was calculated for the final 30 s of each step.
2.4 | Oxygen consumption
Horses wore an open- flow mask on the treadmill through 
which a rheostat- controlled blower drew air. Air flowed 
through 25- cm diameter tubing and across a pneumotacho-
graph (LF- 150B, Vise Medical, Chiba, Japan) connected to 
a differential pressure transducer (TF- 5, Vise Medical) to 
ensure that bias flows during measurements were identi-
cal to those used during calibrations. Bias flow was set to 
keep changes in O2 concentration and CO2 concentrations 
at <1.5% to avoid having the horses rebreathe CO2. Oxygen 
and CO2 concentrations were measured with an O2 and CO2 
analyzer (MG- 360, Vise Medical), and calibrations were 
used to calculate rates of O2 consumption and CO2 produc-
tion with mass flowmeters (CR- 300, Kofloc, Kyoto, Japan) 
using the N2- dilution/CO2- addition mass- balance technique 
(Fedak et al., 1981). Gas analyzer and mass flowmeter out-
puts were also recorded on personal computers using com-
mercial hardware and software (DI- 720 and Windaq Pro+, 
DATAQ, Akron, OH) sampling at 200 Hz.
2.5 | Blood sampling
Before leading a horse onto the treadmill, an 18- gauge cath-
eter (Surflow, Terumo, Tokyo, Japan) was placed in the 
horse's left carotid artery, and an 8- F introducer (MO95H- 8, 
Baxter International, Deerfield, IL) was placed in the right 
jugular vein. A Swan- Ganz catheter (SP5107  U, Becton, 
Dickinson and Company, Franklin Lakes, NJ) was passed via 
the jugular vein so that its tip was positioned in the pulmo-
nary artery, confirmed by measuring pressure at its tip with a 
pressure transducer (P23XL, Becton, Dickinson and Company, 
Franklin Lakes, NJ). Mixed- venous blood samples were drawn 
from the tip of the Swan- Ganz catheter and arterial samples 
from the 18- gauge carotid catheter into heparinized syringes 
at timed intervals for the final 30 s of each step and at 1, 3, 
and 5 min after exhaustion. Samples were stored on ice until 
measured immediately following the experiment. Blood sam-
ples were analyzed with a blood gas analyzer (ABL800 FLEX, 
Radiometer, Copenhagen, Denmark) and O2 saturation (SO2) 
and concentration (CO2) were determined using a hemoximeter 
(ABL80 FLEX- CO- OX, Radiometer, Copenhagen, Denmark). 
Following measurement of blood gases and oximetry, the blood 
was sampled for plasma lactate concentration using a lactate 
analyzer (Biosen S- Line, EKF- diagnostic GmbH, Barleben, 
Germany) after being centrifuged at 1870 × g for 10 min. The 
Swan– Ganz catheter in the pulmonary artery was connected 
to a cardiac output computer (COM- 2, Baxter International, 
Deerfield, IL) so that its thermistor registered pulmonary arte-
rial temperature, could be recorded at each blood sampling and 
used to correct the blood gas measurements.
2.6 | Hypoxic training protocol and 
measurements during exercise in the first 
week of each training period
The procedure for producing the hypoxic condition was 
slightly modified from the method previously described 
4 of 11 |   
MUKAI et Al.
(Ohmura et al., 2010). Briefly, a mixing chamber was con-
nected to the upstream flexible tube on a 25- cm diameter 
open- flow mask through which a flow of N2 was blown 
into the upstream end of the flow system and mixed with a 
bias- flow of air of 80– 120 L/s to create the desired inspired 
O2 concentration. Nitrogen gas flow was controlled with 
a mass flow meter (Model DPM3, Kofloc, Kyoto, Japan) 
connected to compressed gas cylinders through a gas mani-
fold. Nitrogen gas flow was adjusted to maintain 16% or 
18% O2 by monitoring the O2 concentration in the down-
stream arm of the mass flowmeter with an O2 analyzer 
(LC- 240UW, Vise Medical, Chiba, Japan) when horses ran 
in hypoxia.
In the first week of training for all groups, we collected 
arterial blood samples in the final 15 s of cantering at 95% 
̇VO2max during the exercise session to measure arterial blood 
gas variables (ABL800 FLEX and ABL80 FLEX- CO- OX, 
Radiometer, Copenhagen, Denmark) and plasma lactate 
concentration (Biosen S- Line, EKF- diagnostic GmbH, 
Barleben, Germany). We also recorded heart rate (S810, 
Polar, Kempele, Finland) during cantering.
2.7 | Statistical analysis
Data are presented as mean ± standard error (SE). Differences 
in the variables between H16, H18, and N21 during training 
sessions in the first week were analyzed using mixed models 
with a group as a fixed effect and horse as a random effect. 
Post hoc testing was performed by Tukey's test. After train-
ing, the with- in subject changes were analyzed using mixed 
models for differences between groups with a group as a 
fixed effect and horse as a random effect. Tukey's tests were 
used as post hoc tests.
Pearson correlation was used to determine the relation-
ship between the changes in the run time and body weight 
at IET after training and arterial O2 saturation (SaO2), peak 
plasma lactate concentration, and heart rate during exercise 
sessions in the first week of training periods. Statistical anal-
yses were performed with commercial software (JMP 13.1.0, 
SAS Institute Inc, Cary, NC) with significance defined as 
p < 0.05.
3 | RESULTS
3.1 | Blood gas variables, heart rate, and 
plasma lactate concentration during exercise 
sessions in the first week of training
SaO2 was lowest at the last 15 s of a 2- min run at 95% ̇VO2max 
in H16 and highest in N21 (p < 0.0001, Table 1). Arterial O2 
partial pressure (PaO2) in H16 and H18 was lower than that 
in N21 (p < 0.0001, Table 1), and arterial carbon dioxide 
partial pressure (PaCO2) in H16 and H18 was higher than that 
in N21 during exercise (p = 0.0013, Table 1). There were no 
differences in heart rate at the last 15 s of a 2- min run at 95% 
̇VO2max between all groups (p = 0.96, Table 1). Arterial pH of 
H16 was lower than that of N21 (p = 0.0038, Table 1), and 
peak plasma lactate concentration of H16 was higher than 
that of H18 (p = 0.032, Table 1).
3.2 | Effects of normoxic and hypoxic 
training on exercise performance and aerobic 
capacity at IET
After 4 weeks of training, run time (H16, +20.6%, p < 0.0001; 
H18, +11.7%, p = 0.017) and maximal cardiac output ( ̇Qmax
: H16, +8.1%, p = 0.024; H18, +9.5%, p = 0.012) at IET 
increased in H16 and H18, ̇VO2max increased in all groups 
(H16, +9.8%, p = 0.0039; H18, +10.5%, p = 0.0025; N21, 
+8.8%, p = 0.025), and speed at ̇VO2max
(V ̇VO2max
)
 increased 
only in H16 (+7.7%, p = 0.010)(Figure 1, Figure 2 and Table 
2). Blood gas variables including hemoglobin concentration, 
O2 and CO2 partial pressures, and arterial- mixed venous O2 
concentration did not change in all groups during the train-
ing period (Figure 2 and Table 2). Changes in run time and 
V ̇VO2max after 4 weeks of training were different between H16 
and N21 (run time, p = 0.040; V ̇VO2max, p = 0.014), while the 
H16
H18
N21
SaO2 (%)
66.5 ± 1.7 a
74.1 ± 1.7 b
90.9 ± 1.3 c
PaO2 (Torr)
38.8 ± 0.6 a
44.8 ± 2.4 a
68.8 ± 3.3 b
PaCO2 (Torr)
59.1 ± 1.5 a
55.3 ± 3.5 a
42.3 ± 1.3 b
Heart rate (bpm)
203 ± 4 a
202 ± 3 a
202 ± 4 a
Arterial pH
7.210 ± 0.015 a
7.247 ± 0.017 ab
7.281 ± 0.011 b
Peak lactate (mmol/L)
22.3 ± 2.7 a
17.7 ± 1.4 b
18.5 ± 1.0 ab
Arterial O2 saturation (SaO2), arterial O2 partial pressure (PaO2), arterial carbon dioxide partial pressure 
(PaCO2), heart rate, arterial pH, and peak plasma lactate concentration. Values are means ± SE for 7 horses. 
Different letters indicate significant differences between groups (p < 0.05).
TABLE 1 Parameters on aerobic 
capacity and blood gas analysis during the 
exercise session at the 1st week of training
   | 5 of 11
MUKAI et Al.
changes in ̇VO2max, ̇Qmax, SVmax, and blood gas variables were 
not different between the groups ( ̇VO2max, p  =  0.87; ̇Qmax, 
p = 0.74; SVmax, p = 0.99) (Figure 1 and Figure 2). Run time, 
̇VO2max, V ̇VO2max, ̇Qmax, SVmax, and lactate threshold did not 
change after 2 weeks of post- hypoxic training in normoxia in 
all groups compared with those at week 4 (Figure 1 and Table 
FIGURE 1 
Changes in run time (a), 
̇VO2max (b), speed eliciting ̇VO2max (V ̇VO2max
; c) and speed at which plasma lactate 
concentration reached 4 mmol/L (VLA4; 
d) in IET from pre- training to immediate 
post- training (4 weeks) and 2 weeks of 
post- hypoxic training in normoxia (6 weeks) 
either after moderate hypoxia (H16, red), 
mild hypoxia (H18, green), or normoxia 
(N21, blue). Values are mean ±SE. * 
Significant changes from pre- training 
(p < 0.05). † Significant differences 
between groups (p < 0.05)
(a)
(b)
(c)
(d)
FIGURE 2 
Changes in cardiac output 
(a), stroke volume (b), arterial- mixed 
venous O2 difference (c), and hemoglobin 
concentration (d) at exhaustion in IET from 
pre- training to immediate post- training 
(4 weeks) and 2 weeks of post- hypoxic 
training in normoxia (6 weeks) either after 
moderate (H16, red), mild hypoxia (H18, 
green), or normoxia (N21, blue). Values 
are mean ±SE. * Significant changes 
from pre- training (p < 0.05). † Significant 
differences between groups (p < 0.05)
(a)
(b)
(c)
(d)
6 of 11 |   
MUKAI et Al.
TABLE 2 Parameters on exercise performance, aerobic capacity, and blood gas analysis in normoxic incremental exercise tests at week 0, 4, and 6
H16
H18
N21
0 week
4 weeks
6 weeks
0 week
4 weeks
6 weeks
0 week
4 weeks
6 weeks
Run time (s)
416 ± 26
501 ± 36 *
498 ± 36 *
438 ± 25
490 ± 34 *
487 ± 34*
433 ± 18
454 ± 25
464 ± 23
̇VO2max (mL/(min kg))
164 ± 3
180 ± 3 *
180 ± 4 *
161 ± 4
177 ± 3 *
175 ± 6 *
161 ± 4
175 ± 3 *
177 ± 3 *
Body weight (kg)
517 ± 11
493 ± 9 *
494 ± 10 *
518 ± 9
497 ± 11 *
498 ± 12 *
514 ± 10
502 ± 9 *
501 ± 8 *
V ̇VO2max (m/s)
11.1 ± 0.4
12.0 ± 0.4 *
12.0 ± 0.4 *
11.5 ± 0.4
12.1 ± 0.4
12.0 ± 0.5
11.7 ± 0.2
11.6 ± 0.3
11.7 ± 0.3
̇Qmax (mL/(min kg))
666 ± 10
720 ± 19 *
743 ± 19 *
654 ± 16
713 ± 8 *
724 ± 15 *
662 ± 20
703 ± 11
735 ± 16 *
SVmax (mL/kg)
3.20 ± 0.08
3.39 ± 0.12
3.51 ± 0.13 *
3.13 ± 0.09
3.35 ± 0.07 *
3.42 ± 0.10 *
3.18 ± 0.13
3.32 ± 0.10
3.48 ± 0.14 *
HRmax (bpm)
209 ± 5
215 ± 5
213 ± 3
209 ± 4
213 ± 3
212 ± 4
209 ± 5
213 ± 4
214 ± 4
VHRmax (m/s)
10.2 ± 0.4
10.9 ± 0.5
11.0 ± 0.4
10.4 ± 0.5
11.0 ± 0.4
11.1 ± 0.4
10.5 ± 0.3
10.6 ± 0.4
10.7 ± 0.4
Ca- vO2 (mL/dL)
24.6 ± 0.2
24.6 ± 0.2
24.2 ± 0.2
24.8 ± 0.2
24.8 ± 0.2
24.1 ± 0.2
24.3 ± 0.1
24.8 ± 0.2
24.1 ± 0.2
[Hb] (g/dL)
23.9 ± 0.5
23.7 ± 0.6
23.6 ± 0.6
24.0 ± 0.4
24.1 ± 0.5
23.7 ± 0.5
23.5 ± 0.5
23.8 ± 0.4
23.5 ± 0.6
PaO2 (Torr)
78.8 ± 0.7
79.0 ± 0.5
80.4 ± 1.0
83.0 ± 1.0
81.0 ± 1.0
79.2 ± 1.2
84.0 ± 0.9
79.8 ± 1.0
79.2 ± 0.8
PvO2 (Torr)
21.2 ± 0.1
19.7 ± 0.3
20.6 ± 0.3
22.3 ± 0.2
20.8 ± 0.2
21.0 ± 0.3
22.4 ± 0.2
19.9 ± 0.2
20.4 ± 0.3
PaCO2 (Torr)
52.9 ± 0.5
55.8 ± 0.6
55.0 ± 0.8
53.1 ± 0.6
56.7 ± 0.6
56.6 ± 0.6
52.9 ± 0.4
54.4 ± 0.6
54.7 ± 0.5
PvCO2 (Torr)
112.2 ± 1.6
126.7 ± 2.9
122.2 ± 2.2
110.9 ± 1.7
121.3 ± 2.2
120.0 ± 2.2
114.2 ± 1.7
119.5 ± 2.6
122.1 ± 2.2
SaO2 (%)
87.7 ± 0.4
86.5 ± 0.4
86.5 ± 0.5
88.9 ± 0.4
86.8 ± 0.5
85.9 ± 0.5
89.5 ± 0.4
87.5 ± 0.5
86.7 ± 0.4
SvO2 (%)
13.5 ± 0.3
11.7 ± 0.6
11.7 ± 0.4
14.9 ± 0.4
12.7 ± 0.5
12.5 ± 0.6
14.7 ± 0.5
12.5 ± 0.5
12.0 ± 0.5
pHa
7.207 ± 0.005
7.200 ± 0.009
7.203 ± 0.008
7.235 ± 0.006
7.218 ± 0.009
7.213 ± 0.010
7.227 ± 0.007
7.215 ± 0.010
7.220 ± 0.009
pHv
7.084 ± 0.006
7.070 ± 0.011
7.073 ± 0.008
7.101 ± 0.007
7.094 ± 0.010
7.073 ± 0.009
7.097 ± 0.011
7.085 ± 0.011
7.087 ± 0.009
Run time, maximal oxygen consumption ( ̇VO2max), body weight, speed eliciting ̇VO2max (V ̇VO2max), cardiac output ( ̇Qmax), cardiac stroke volume (SVmax), maximal heart rate (HRmax), speed eliciting HRmax (VHRmax), arterial and 
mixed- venous O2 difference (Ca- vO2), arterial and mixed- venous O2 partial pressure (PaO2, PvO2), arterial and mixed- venous carbon dioxide partial pressure (PaCO2, PvCO2), hemoglobin concentration ([Hb]), arterial and mixed 
venous O2 saturation (SaO2, SvO2), and arterial and mixed venous pH (pHa, pHv) at exhaustion during normoxic incremental exercise tests. Values are means ±SE for seven horses. *Significant changes from 0 week (p < 0.05).
   | 7 of 11
MUKAI et Al.
2). Body weight decreased after 4 weeks of training in all 
groups (H16, −4.7%, p < 0.0001; H18, −4.1%, p < 0.0001; 
N21, −2.4%, p = 0.0003) and there was a significantly greater 
weight loss in H16 compared to N21 (p = 0.021), but not be-
tween H18 and N21. These reductions in body weight lasted 
for 2 weeks after a switch to normoxic training (Table 2).
3.3 | Correlations between the variables 
during the exercise session and the changes of 
variables at IET after 4 weeks of training
There were significant correlations between SaO2 during ex-
ercise and the changes in run time (r = −0.59, p = 0.0067; 
Figure 3a), between peak plasma lactate concentration during 
exercise and the changes in run time (r = 0.66, p = 0.0017; 
Figure 3b), and between SaO2 during exercise and the changes 
in body weight (r = 0.61, p = 0.0040; Figure 3d). No sig-
nificant correlations were observed between heart rate during 
exercise and the changes in run time (r = −0.077, p = 0.75; 
Figure 3c).
4 | DISCUSSION
The purpose of this study was to determine whether high- 
intensity training in moderate and mild hypoxia could improve 
exercise performance and aerobic capacity to a greater extent 
than the same training in normoxia. In addition, we sought to 
determine if two weeks of post- hypoxic training in normoxia 
could maintain the benefit of hypoxic training. First, we dem-
onstrated that horses trained in 16% O2 enhanced run time 
and V ̇VO2max at IET more than horses trained in normoxia and 
that horses trained in 18% O2 showed a similar adaptation as 
H16, but there was no statistical significance between H18 
and N21. In addition, the acquired hypoxic training effects 
on performance and aerobic capacity were sustained after 
2 weeks of post- hypoxic training in normoxia.
4.1 | Training effects on exercise 
performance and aerobic capacity after 
hypoxic training
Despite several human studies reported no additional ben-
efits in LLTH training (Faiss et al., 2013), 4 weeks of hy-
poxic training in H16 showed greater improvements in run 
time and V ̇VO2max at IET than normoxic training. Vogt and 
Hoppeler (2010) stated that there is no clear trend in the ef-
fects of LLTH training on performance at sea level and no 
conclusive recommendations can be made as to which al-
titude, exposure duration, and exercise intensity might be 
beneficial. In contrast, (McLean et al., 2014) indicated that 
enhancements in normoxic performance appear most likely 
FIGURE 3 
Correlations between SaO2 
(a), peak plasma lactate concentration (b), 
and heart rate (c) during exercise session in 
the first week and the change in run time at 
IET after 4 weeks of training, and between 
SaO2 during exercise session and the change 
in body weight (d) at IET after 4 weeks of 
training. Red, green, and blue dots indicate 
moderate hypoxia (H16), mild hypoxia 
(H18), and normoxia (N21), respectively. 
Solid lines indicate regression lines and pink 
areas indicate 95% confidence intervals.
(a)
(b)
(c)
(d)
8 of 11 |   
MUKAI et Al.
following high- intensity and short- term training in hypoxia. 
Despite our hypoxic settings (FIO2: 16% and 18%) being con-
sidered as moderate and/or mild condition for human hypoxic 
training, horses exercised in H16 and H18 experienced se-
vere arterial hypoxemia in this study, and their end- exercise 
SaO2 declined to 66.5 ± 1.7% and 74.1 ± 1.7%, respectively 
(Table 1). Thoroughbred horses often exhibit arterial hypox-
emia during high- intensity exercise even in normoxia mostly 
due to diffusion limitations in the lungs (Wagner et al., 1989). 
Previous literature has demonstrated that exercise- induced 
arterial hypoxemia also occurs in highly- trained human ath-
letes during heavy exercise in normoxia and hypoxia, and 
the end- exercise SaO2 at FIO2 of 21% was similar to that ob-
served in horses (91 ± 1%), while SaO2 at FIO2 of 17% was 
not as low as that observed horses (83 ± 1%) (Vogiatzis et al., 
2007). These findings suggest that hypoxia may cause more 
severe exercise- induced arterial hypoxemia in horses than in 
humans, and these differences between horses and humans 
in the severity of exercise- induced arterial hypoxemia during 
hypoxic training might induce different training adaptations 
on performance and aerobic capacity.
In equine studies, whereas (Davie et al., 2017) reported no 
additional improvements in heart rate and blood lactate con-
centration during incremental treadmill tests after 6 weeks 
of moderate- intensity hypoxic training (3 hypoxic and 3 nor-
moxic sessions/week, total 30 min/session, 15% inspired O2), 
Ohmura et al. (2017) demonstrated that all- out running for 
2– 3 min in hypoxia (15.1% inspired O2) twice a week for 
3 weeks increased ̇VO2max of well- trained horses in normoxia. 
Our previous study in horses also showed that 4 weeks of 
high- intensity training in hypoxia (100% ̇VO2max 2 min, 3 ses-
sions/week, 15% inspired O2) improved performance, ̇VO2max
, and maximal cardiac output to a greater extent than nor-
moxic training (Mukai et al., 2020). Training programs var-
ied among these studies, including intensity and duration of 
training, training status of horses (untrained or trained), and 
hypoxic exposure duration, but these interventions used sim-
ilar O2 concentrations of hypoxic gas. Given that the program 
of Davie's group, which used a longer duration of training 
and hypoxic exposure, but lower training intensity, showed 
no benefit in hypoxic training. The key factors for hypoxic 
training adaptations in horses may also be high- intensity and 
short- term training as McLean et al. (2014) suggested.
The changes in V ̇VO2max in all groups were very similar 
to those in run time at IET in our study (Figure 1). While 
V ̇VO2max is not a direct parameter for running economy, Billat 
et al. (2003) reported that V ̇VO2max is highly correlated with 
10- km performance time (r = −0.86 in men, r = −0.95 in 
women) and V ̇VO2max predicts performance better than ̇VO2max 
since V ̇VO2max integrates the energy cost of running in addition 
to ̇VO2max. Given that the changes in ̇VO2max after 4 weeks of 
training were similar in all groups in this study, the improve-
ments in V ̇VO2max may reflect enhanced running economy in 
submaximal exercise at IET. Several researchers also demon-
strated that hypoxic training improves the running economy 
compared with normoxic training in humans (Katayama 
et al., 2003; Park et al., 2018; Saunders et al., 2004; Sinex 
& Chapman, 2015). Barnes and Kilding (2015) stated that 
altitude acclimatization induces both central and peripheral 
adaptations that improve oxygen delivery and utilization, 
mechanisms that may improve running economy. In contrast, 
Saunders et al. (2004) suggested that the lower aerobic cost 
of running is not related to ventilation, heart rate, respiratory 
exchange ratio, or hemoglobin mass. These conflicting re-
sults indicate that the mechanism of improved running econ-
omy after hypoxic training is unclear and further studies are 
needed.
̇VO2max, ̇Qmax, and SVmax at IET increased similarly in all 
groups throughout the study. In our previous study (Mukai 
et al., 2020), however, we observed greater ̇VO2max, ̇Qmax, and 
SVmax in the hypoxic group compared to that of the normoxic 
group in a similar study design. The causes for these differ-
ences are not clear, but the minor differences in the training 
intensity (100% ̇VO2max vs. 95% ̇VO2max), the degree of hy-
poxia (15% O2 vs. 16% or 18% O2), and the age of horses 
(6.5 years vs. 7.9 years) might affect training adaptation on 
aerobic capacity.
On the other hand, Ca- vO2 was unchanged during the 
training period in all groups, indicating that the consumed O2 
in working muscle, that is mitochondrial oxidative capacity, 
did not change after training. Therefore, the majority of the 
increase in ̇VO2maxseems to be induced by the increase in O2 
delivery. These results suggest that the mitochondrial oxida-
tive capacity is not a limiting factor of ̇VO2maxin horses and 
O2 delivery is implicated as the primary limitation for ̇VO2max
, as previously described (Jones & Lindstedt, 1993).
4.2 | Correlations between SaO2 during 
exercise session and the changes of variables at 
IET after 4 weeks of training
We observed a moderate negative correlation (r  =  −0.59) 
between SaO2 during the training session and the increase 
in run time at IET, and also a moderate positive correla-
tion (r = 0.61) between SaO2 and body weight loss (Figure 
3), which suggests that a greater reduction in SaO2 during 
the exercise session induces a greater improvement in per-
formance and greater weight loss after 4 weeks of training. 
Given that the lower SaO2 during the training session could 
simultaneously induce both positive and negative effects, 
trainers should understand the possibility of hypoxia- induced 
weight loss and set an optimal training program for peak 
racing performance. As monitoring each horse's SaO2 dur-
ing exercise is not practical at the training track, we recom-
mend monitoring peak lactate concentration instead, which 
   | 9 of 11
MUKAI et Al.
correlated moderately with the change in run time after train-
ing (r = 0.66), as well as SaO2 (Figure 3).
4.3 | Effect of hypoxic training on body 
weight loss
At the same absolute exercise intensity, exercise in hypoxia 
is perceived as harder (i.e., lower SaO2 and/or higher lactate 
concentration) and the relative exercise intensity is higher in 
hypoxia due to the lower ̇VO2maxthan in normoxia (Ohmura 
et al., 2020). Consequently, hypoxic training can lead to in-
creased energy expenditure, decreased energy intake, and 
greater body weight loss compared to normoxic training at 
the same absolute intensity. Furthermore, Katayama et al. 
(2010) reported that carbohydrate utilization increased dur-
ing exercise and recovery period in moderate hypoxia com-
pared with normoxic exercise at the same relative intensity. 
These findings suggest that a shift in substrate utilization 
may also occur during hypoxic training in horses. Contrary 
to these results, our previous study demonstrated that well- 
trained horses did not reduce their body weight after 3 weeks 
of high- intensity training (5 sessions/3  weeks) in hypoxia 
(Ohmura et al., 2017). These contradictory data indicate that 
the training status at the beginning of training, as well as the 
intensity, frequency, and volume of training, may affect the 
extent of body weight loss during hypoxic training. However, 
the mechanism of body weight loss during hypoxic training 
is still unclear and further studies are needed to investigate 
the relationship between hypoxic training and body weight 
loss.
4.4 | Hematological changes with 
hypoxic training
Consistent with human studies (Roels et al., 2005; Truijens 
et al., 2003) and our previous study (Mukai et al., 2020), 
hemoglobin concentrations both at rest and at exhaustion in 
all groups did not increase after training in our present study. 
While LHTH and/or LHTL training usually aims to enhance 
athletic performance by stimulating an increase in serum 
erythropoietin and erythrocyte volume, only a few well- 
controlled LLTH studies on trained or elite athletes have 
reported increments in hemoglobin concentration (Bonetti 
et al., 2006), and none have reported any increases in eryth-
rocyte volume and/or hemoglobin mass. Some  studies 
showed that intermittent hypoxic exposure at rest (3 h/day, 
5 days/week for 4 weeks at 4000– 5500 m altitude) increases 
serum erythropoietin only immediately after a 3 h hypoxic 
exposure, but no significant differences were observed in 
erythrocyte volume or hemoglobin mass compared with the 
normoxic control (Abellán et al., 2005; Gore et al., 2006). 
Millet et al. (2010) reported that the minimum daily dose for 
stimulating erythropoiesis seems to be 12 h/day. These previ-
ous reports in humans suggest that the exposure duration to 
hypoxia (approximately 3 min/session) in this study was too 
short to increase erythrocyte volume and hemoglobin mass/
concentration.
4.5 | Maintenance of post- hypoxic 
training effects
There is contradictory evidence concerning how long the ac-
quired benefits of hypoxic training last after a return to sea 
level. Pottgiesser et al. (2012) reported that nearly hypoxia- 
induced hematological changes observed after 4  weeks of 
LHTL training may be lost within 9 days, while Brocherie 
et al. (2015) showed that 14 days of hypoxic training im-
proved repeated sprint performance and hemoglobin mass, 
with the benefits lasting for at least 3 weeks post- intervention. 
Another study also reported that the increase in total hemo-
globin mass of elite runners at altitude training camp is stable 
for 14 days after returning to sea level (Prommer et al., 2010). 
Consistent with the findings of Brocherie et al. (2015), exer-
cise performance and aerobic capacity after 2 weeks of post- 
hypoxic training in normoxia were very similar to those after 
4 weeks of hypoxic training in our study, which indicates 
that most adaptations induced by hypoxic training are main-
tained after 2 weeks of normoxic training. LLTH training in 
our study did not induce any changes in hemoglobin concen-
tration throughout the study, which suggests that horses did 
not gain or lose any benefits from hematological adaptations. 
This may be one of the reasons that horses maintained their 
performance and aerobic capacity after post- hypoxic training 
in normoxia.
4.6 | Experimental design of this study
Our study design altered two factors: the ̇VO2- based relative 
training intensity and FIO2. In some human studies, subjects 
trained at matched relative intensity either in normoxia or hy-
poxia experience similar adaptations after training (McLean 
et al., 2014; Truijens et al., 2003). In our design, horses 
trained in moderate hypoxia showed a greater adaptation in 
performance without increasing absolute speed. Given that 
Thoroughbred horses often experience musculoskeletal in-
juries, we consider that this model of hypoxic training may 
have benefits of no additional speed/mechanical load. Even 
if we match the relative training intensity in both normoxia 
and hypoxia, the absolute speed or mechanical load will 
decrease in the hypoxic training program, which indicates 
that we are changing two factors again: the absolute speed/ 
mechanical load and FIO2. In addition, we were concerned 
10 of 11 |   
MUKAI et Al.
about performing two incremental exercise tests for pre- 
training measurements in normoxia and for the relative in-
tensity in hypoxia during a relatively short period. Indeed, we 
understand that further studies that match the relative training 
intensity in normoxia and hypoxia are needed to clarify the 
mechanism of hypoxic training in horses.
5 | CONCLUSION
In this study, we demonstrated that 4  weeks of training in 
moderate (H16), but not mild hypoxia (H18) was sufficient to 
elicit greater improvements in performance and running econ-
omy than normoxic training and that the effects of the hypoxic 
training were maintained over 2 weeks of post- hypoxic train-
ing. Although trainers should monitor weight loss, hypoxic 
training may be a strategic option for an equine training pro-
gram without increasing locomotory mechanical stress.
ACKNOWLEDGMENTS
The authors thank the technical staff of the JRA Equine 
Research Institute for expert technical assistance, training, 
and husbandry throughout the study.
CONFLICT OF INTEREST
This study was funded by the Japan Racing Association. 
KM, HO, YT, and TT are employees of the Japan Racing 
Association.
AUTHOR CONTRIBUTIONS
Conceptualization: KM, HO, YK, and TT. Investigation: 
KM, HO, YT, and TT. Formal analysis: KM and TT. 
Methodology: KM, HO, and TT. Writing— original draft: 
KM. Writing— review & editing: KM, HO, YT, and YK.
ORCID
Kazutaka Mukai 
 https://orcid.org/0000-0002-1992-6634 
Yuji Takahashi 
 https://orcid.org/0000-0002-2139-6142 
Yu Kitaoka 
 https://orcid.org/0000-0001-6932-2735 
REFERENCES
Abellán, R., Remacha, A. F., Ventura, R., Sardà, M. P., Segura, J., & 
Rodríguez, F. A. (2005). Hematologic response to four weeks 
of intermittent hypobaric hypoxia in highly trained athletes. 
Haematologica, 90(1), 126– 127.
Barnes, K. R., & Kilding, A. E. (2015). Strategies to improve running 
economy. Sports Medicine (Auckland, N. Z.), 45(1), 37– 56.
Billat, V., Lepretre, P. M., Heugas, A. M., Laurence, M. H., Salim, D., & 
Koralsztein, J. P. (2003). Training and bioenergetic characteristics 
in elite male and female Kenyan runners. Medicine and Science in 
Sports and Exercise, 35(2), pp. 297– 304; discussion 5– 6.
Bonetti, D. L., & Hopkins, W. G. (2009). Sea- level exercise perfor-
mance following adaptation to hypoxia: a meta- analysis. Sports 
Medicine (Auckland, N. Z.), 39(2), 107– 127.
Bonetti, D. L., Hopkins, W. G., & Kilding, A. E. (2006). High- intensity 
kayak performance after adaptation to intermittent hypoxia. 
International Journal of Sports Physiology and Performance, 1(3), 
246– 260.
Brocherie, F., Millet, G. P., Hauser, A., Steiner, T., Rysman, J., Wehrlin, 
J. P., & Girard, O. (2015). "Live High- Train Low and High" 
Hypoxic Training Improves Team- Sport Performance. Medicine 
and Science in Sports and Exercise, 47(10), 2140– 2149.
Czuba, M., Waskiewicz, Z., Zajac, A., Poprzecki, S., Cholewa, J., & 
Roczniok, R. (2011). The effects of intermittent hypoxic train-
ing on aerobic capacity and endurance performance in cyclists. 
Journal of Sports Science and Medicine, 10(1), 175– 183.
Davie, A. J., Evans, D. L., Hodgson, D. R., & Rose, R. J. (1999). Effects 
of muscle glycogen depletion on some metabolic and physiologi-
cal responses to submaximal treadmill exercise. Canadian Journal 
of Veterinary Research, 63(4), 241– 247.
Davie, A. J., Wen, L., Cust, A. R. E., Beavers, R., Fyfe, T., & Zhou, 
S. (2017). The effects of moderate intensity training in a hypoxic 
environment on transcriptional responses in Thoroughbred horses. 
Biology Open, 6(7), 1035– 1040.
Desplanches, D., Hoppeler, H., Linossier, M. T., Denis, C., Claassen, 
H., Dormois, D., Lacour, J. R., & Geyssant, A. (1993). Effects of 
training in normoxia and normobaric hypoxia on human muscle 
ultrastructure. Pflugers Archiv European Journal of Physiology, 
425(3– 4), 263– 267.
Dick, F. W. (1992). Training at altitude in practice. International Journal 
of Sports Medicine, 13(Suppl 1), S203– S206.
Eaton, M. D., Evans, D. L., Hodgson, D. R., & Rose, R. J. (1995). 
Maximal accumulated oxygen deficit in thoroughbred horses. 
Journal of Applied Physiology, 78(4), 1564– 1568.
Faiss, R., Girard, O., & Millet, G. P. (2013). Advancing hypoxic train-
ing in team sports: from intermittent hypoxic training to repeated 
sprint training in hypoxia. British Journal of Sports Medicine, 
47(Suppl 1), i45– 50.
Fedak, M. A., Rome, L., & Seeherman, H. J. (1981). One- step N₂- 
dilution technique for calibrating open- circuit VO₂ measuring sys-
tems. Journal of Applied Physiology, 51(3), 772– 776.
Gore, C. J., Hahn, A. G., Aughey, R. J., Martin, D. T., Ashenden, M. 
J., Clark, S. A., Garnham, A. P., Roberts, A. D., Slater, G. J., & 
Mckenna, M. J. (2001). Live high:train low increases muscle buf-
fer capacity and submaximal cycling efficiency. Acta Physiologica 
Scandinavica, 173(3), 275– 286.
Gore, C. J., Rodriguez, F. A., Truijens, M. J., Townsend, N. E., 
 Stray- Gundersen, J., & Levine, B. D. (2006). Increased serum 
erythropoietin but not red cell production after 4 wk of inter-
mittent hypobaric hypoxia (4,000– 5,500 m). Journal of Applied 
Physiology, 101(5), 1386– 1393.
Hamlin, M. J., Marshall, H. C., Hellemans, J., Ainslie, P. N., & Anglem, 
N. (2010). Effect of intermittent hypoxic training on 20 km time 
trial and 30 s anaerobic performance. Scandinavian Journal of 
Medicine and Science in Sports, 20(4), 651– 661.
Hendriksen, I. J., & Meeuwsen, T. (2003). The effect of intermittent 
training in hypobaric hypoxia on sea- level exercise: a cross- 
over study in humans. European Journal of Applied Physiology, 
88(4– 5), 396– 403.
Jones, J. H., & Lindstedt, S. L. (1993). Limits to maximal performance. 
Annual Review of Physiology, 55, 547– 569.
Katayama, K., Goto, K., Ishida, K., & Ogita, F. (2010). Substrate utiliza-
tion during exercise and recovery at moderate altitude. Metabolism, 
59(7), 959– 966.
   | 11 of 11
MUKAI et Al.
Katayama, K., Matsuo, H., Ishida, K., Mori, S., & Miyamura, M. 
(2003). Intermittent hypoxia improves endurance performance 
and submaximal exercise efficiency. High Altitude Medicine & 
Biology, 4(3), 291– 304.
Kitaoka, Y., Endo, Y., Mukai, K., Aida, H., Hiraga, A., & Hatta, H. 
(2014). Muscle glycogen breakdown and lactate metabolism 
during intensive exercise in Thoroughbred horses. The Journal of 
Physical Fitness and Sports Medicine, 3(4), 451– 456.
McLean, B. D., Gore, C. J., & Kemp, J. (2014). Application of ‘live 
low- train high’ for enhancing normoxic exercise performance in 
team sport athletes. Sports Medicine (Auckland, N. Z.), 44(9), 
1275– 1287.
Millet, G. P., Roels, B., Schmitt, L., Woorons, X., & Richalet, J. P. 
(2010). Combining hypoxic methods for peak performance. Sports 
Medicine (Auckland, N. Z.), 40(1), 1– 25.
Mukai, K., Hiraga, A., Takahashi, T., Matsui, A., Ohmura, H., Aida, 
H., & Jones, James H. (2017). Effects of maintaining different 
exercise intensities during detraining on aerobic capacity in 
Thoroughbreds. American Journal of Veterinary Research, 78(2), 
215– 222.
Mukai, K., Ohmura, H., Matsui, A., Aida, H., Takahashi, T., & Jones, 
J. (2020). High- intensity training in normobaric hypoxia enhances 
exercise performance and aerobic capacity in Thoroughbred 
horses: A randomized crossover study. Physiological Reports, 
8(10), e14442.
Ohmura, H., Mukai, K., Matsui, A., Takahashi, T., & Jones, J. H. (2020). 
Cardiopulmonary function during supramaximal exercise in hy-
poxia, normoxia and hyperoxia in Thoroughbred horses. Journal 
of Equine Science, 31(4), 67– 73.
Ohmura, H., Mukai, K., Takahashi, T., Matsui, A., Hiraga, A., & Jones, 
J. H. (2010). Comparison of net anaerobic energy utilisation esti-
mated by plasma lactate accumulation rate and accumulated oxy-
gen deficit in Thoroughbred horses. Equine Veterinary Journal, 
42(Suppl 38), 62– 69.
Ohmura, H., Mukai, K., Takahashi, Y., Takahashi, T., & Jones, J. H. 
(2017). Hypoxic training increases maximal oxygen consump-
tion in Thoroughbred horses well- trained in normoxia. Journal of 
Equine Science, 28(2), 41– 45.
Park, H. Y., Shin, C., & Lim, K. (2018). Intermittent hypoxic training 
for 6 weeks in 3000 m hypobaric hypoxia conditions enhances ex-
ercise economy and aerobic exercise performance in moderately 
trained swimmers. Biology of sport, 35(1), 49– 56.
Pascoe, J. R., Hiraga, A., Hobo, S., Birks, E. K., Yarbrough, T. B., 
Takahashi, T., Hada, T., Aida, H., Steffey, E. P., & Jones, J. H. 
(1999). Cardiac output measurements using sonomicrometer crys-
tals on the left ventricle at rest and exercise. Equine Veterinary 
Journal. Supplement, 30, 148– 152.
Pottgiesser, T., Garvican, L. A., Martin, D. T., Featonby, J. M., Gore, 
C. J., & Schumacher, Y. O. (2012). Short- term hematological 
effects upon completion of a four- week simulated altitude camp. 
International Journal of Sports Physiology and Performance, 7(1), 
79– 83.
Prommer, N., Thoma, S., Quecke, L., Gutekunst, T., Völzke, C., 
Wachsmuth, N., Niess, A. M., & Schmidt, W. (2010). Total hemo-
globin mass and blood volume of elite Kenyan runners. Medicine 
and Science in Sports and Exercise, 42(4), 791– 797.
Puype, J., Van Proeyen, K., Raymackers, J. M., Deldicque, L., & Hespel, 
P. (2013). Sprint interval training in hypoxia stimulates glycolytic 
enzyme activity. Medicine and Science in Sports and Exercise, 
45(11), 2166– 2174.
Robertson, E. Y., Saunders, P. U., Pyne, D. B., Aughey, R. J., Anson, J. 
M., & Gore, C. J. (2010). Reproducibility of performance changes 
to simulated live high/train low altitude. Medicine and Science in 
Sports and Exercise, 42(2), 394– 401.
Roels, B., Millet, G. P., Marcoux, C. J., Coste, O., Bentley, D. J., & 
Candau, R. B. (2005). Effects of hypoxic interval training on cy-
cling performance. Medicine and Science in Sports and Exercise, 
37(1), 138– 146.
Saunders, P. U., Telford, R. D., Pyne, D. B., Cunningham, R. B., Gore, 
C. J., Hahn, A. G., & Hawley, J. A. (2004). Improved running 
economy in elite runners after 20 days of simulated moderate- 
altitude exposure. Journal of Applied Physiology, 96(3), 931– 937.
Sinex, J. A., & Chapman, R. F. (2015). Hypoxic training methods for 
improving endurance exercise performance. Journal of Sport and 
Health Science, 4(4), 325– 332.
Truijens, M. J., Toussaint, H. M., Dow, J., & Levine, B. D. (2003). 
Effect of high- intensity hypoxic training on sea- level swimming 
performances. Journal of Applied Physiology, 94(2), 733– 743.
Vogiatzis, I., Georgiadou, O., Koskolou, M., Athanasopoulos, D., 
Kostikas, K., Golemati, S., Wagner, H., Roussos, C., Wagner, P. 
D., & Zakynthinos, S. (2007). Effects of hypoxia on diaphragmatic 
fatigue in highly trained athletes. Journal of Physiology, 581(Pt 
1), 299– 308.
Vogt, M., & Hoppeler, H. (2010). Is hypoxia training good for muscles 
and exercise performance? Progress in Cardiovascular Diseases, 
52(6), 525– 533.
Vogt, M., Puntschart, A., Geiser, J., Zuleger, C., Billeter, R., & Hoppeler, 
H. (2001). Molecular adaptations in human skeletal muscle to en-
durance training under simulated hypoxic conditions. Journal of 
Applied Physiology, 91(1), 173– 182.
Wagner, P. D., Gillespie, J. R., Landgren, G. L., Fedde, M. R., Jones, 
B. W., DeBowes, R. M., Pieschl, R. l., & Erickson, H. H. (1989). 
Mechanism of exercise- induced hypoxemia in horses. Journal of 
Applied Physiology, 66(3), 1227– 1233.
How to cite this article: Mukai K, Ohmura H, 
Takahashi Y, Kitaoka Y, Takahashi T. Four weeks of 
high- intensity training in moderate, but not mild 
hypoxia improves performance and running economy 
more than normoxic training in horses. Physiol Rep. 
2021;9:e14760. https://doi.org/10.14814/phy2.14760

Four weeks of high-intensity training in moderate, but not mild hypoxia improves performance and running economy more than normoxic training in horses.
Mukai, Kazutaka,Ohmura, Hajime,Takahashi, Yuji,Kitaoka, Yu,Takahashi, Toshiyuki