Live high:train low increases muscle buffer capacity and submaximal cycling efficiency

Abstract

This study investigated whether hypoxic exposure increased muscle buffer capacity (beta(m)) and mechanical efficiency during exercise in male athletes. A control (CON, n=7) and a live high:train low group (LHTL, n=6) trained at near sea level (600 m), with the LHTL group sleeping for 23 nights in simulated moderate altitude (3000 m). Whole body oxygen consumption (VO2) was measured under normoxia before, during and after 23 nights of sleeping in hypoxia, during cycle ergometry comprising 4 x 4-min submaximal stages, 2-min at 5.6 +/- 0.4 W kg(-1), and 2-min 'all-out' to determine total work and VO(2peak). A vastus lateralis muscle biopsy was taken at rest and after a standardized 2-min 5.6 +/- 0.4 W kg(-1) bout, before and after LHTL, and analysed for beta(m) and metabolites. After LHTL, beta(m) was increased (18%, P < 0.05). Although work was maintained, VO(2peak) fell after LHTL (7%, P < 0.05). Submaximal VO2 was reduced (4.4%, P < 0.05) and efficiency improved (0.8%, P < 0.05) after LHTL probably because of a shift in fuel utilization. This is the first study to show that hypoxic exposure, per se, increases muscle buffer capacity. Further, reduced VO2 during normoxic exercise after LHTL suggests that improved exercise efficiency is a fundamental adaptation to LHTL.

Full text

Live high:train low increases muscle buffer capacity
and submaximal cycling ef®ciency
C. J. GORE,
1
A. G. HAHN,
2
R. J. AUGHEY,
3
D. T. MARTIN,
2
M. J. ASHENDEN,
2
S. A. CLARK,
2
A. P. GARNHAM,
4
A. D. ROBERTS,
5
G. J. SLATER
2
and M. J. MCKENNA
3
1 Australian Institute of Sport, Adelaide, Australia
2 Department of Physiology, Australian Institute of Sport, Canberra, Australia
3 School of Human Movement, Recreation and Performance, Centre for Rehabilitation, Exercise and Sports Science, Victoria
University of Technology, Melbourne, Australia
4 School of Health Sciences, Deakin University, Melbourne, Australia
5 Centre of Sports Studies, Canberra University, Canberra, Australia
ABSTRACT
This study investigated whether hypoxic exposure increased muscle buffer capacity (bm) and
mechanical ef®ciency during exercise in male athletes. A control (CON, n7) and a live high:train
low group (LHTL, n6) trained at near sea level (600 m), with the LHTL group sleeping for 23 nights
in simulated moderate altitude (3000 m). Whole body oxygen consumption ( _
V
O
2
) was measured
under normoxia before, during and after 23 nights of sleeping in hypoxia, during cycle ergometry
comprising 4 4-min submaximal stages, 2-min at 5.6  0.4 W kg
±1
, and 2-min `all-out' to determine
total work and _
V
O
2peak
. A vastus lateralis muscle biopsy was taken at rest and after a standardized 2-
min 5.6  0.4 W kg
±1
bout, before and after LHTL, and analysed for bm and metabolites. After LHTL,
bm was increased (18%, P< 0.05). Although work was maintained, _
V
O
2peak
fell after LHTL (7%,
P< 0.05). Submaximal _
V
O
2
was reduced (4.4%, P< 0.05) and ef®ciency improved (0.8%, P< 0.05)
after LHTL probably because of a shift in fuel utilization. This is the ®rst study to show that hypoxic
exposure, per se, increases muscle buffer capacity. Further, reduced _
V
O
2
during normoxic exercise
after LHTL suggests that improved exercise ef®ciency is a fundamental adaptation to LHTL.
Keywords altitude training, cycling ef®ciency, hypoxia, muscle buffering.
Received 8 December 2000, accepted 29 May 2001
Altitude training for improved performance at sea
level remains highly contentious (Rusko 1996, Saltin
1996, Wolski et al. 1996). In part, this may be a
consequence of any performance change being small
and variable between individuals (Rusko 1996).
Recently an alternative approach to enhance athletic
performance has been mooted, where athletes live at
moderate altitude and train near sea level. This
method of using hypobaric hypoxia improved the sea-
level 5000 or 3000 m run time in both college (Levine
& Stray-Gundersen 1997) and elite level runners
(Stray-Gundersen et al. 2001), but enhanced perform-
ance is a relatively rare outcome among those studies
of altitude training that have used a control (CON)
group. Because many countries lack suitable geo-
graphy, the so-called `live high:train low' (LHTL)
approach (Levine & Stray-Gundersen 1997) has been
further re®ned to include living at simulated altitude
under normobaric conditions (Rusko 1996).
Regardless of whether LHTL or natural altitude
sojourns are used by athletes there is some evidence to
challenge the traditional paradigm that the key adapta-
tion for any performance bene®t is increased red cell
mass (Mairba
Èurl 1994) and the concomitant increase in
maximal aerobic power ( _
V
O
2max
) that has otherwise
been associated with polycythaemia (Buick et al. 1980).
Two studies have reported that training at altitude
(2000±2700 m) induced a 5±6% increase in skeletal
muscle in-vitro buffer capacity (bm) (Mizuno et al. 1990,
Saltin et al. 1995a). Furthermore, a carefully conducted
study has recently reported a signi®cant (5%)
improvement in the net mechanical ef®ciency of sub-
maximal cycling subsequent to a 21-day mountain
ascent (6194 m) (Green et al. 2000b). The mechanism
Correspondence: C. J. Gore, Australian Institute of Sport ± Adelaide, PO Box 21, Henley Beach, Adelaide 5022, South Australia, Australia.
Acta Physiol Scand 2001, 173, 275±286
Ó2001 Scandinavian Physiological Society 275

of increased bm and mechanical ef®ciency is unclear,
but in both cases hypoxia is a likely candidate.
Given the potential importance of anaerobic meta-
bolism (Bulbulian et al. 1986) and ef®ciency (Snell
& Mitchell 1984) to performance, even in highly trained
endurance athletes, further investigation of possible
anaerobic adaptations to hypoxia is clearly warranted.
Based on the reported effect of 2 weeks living and
training at natural altitude (Saltin et al. 1995a), we
hypothesized that merely sleeping in moderate hypoxia
(LHTL) for suf®cient duration would improve bm.
Secondly, based on the observation of Green et al.
(2000b), we hypothesized that LHTL of suf®cient
duration would improve gross mechanical ef®ciency
during submaximal cycle ergometry conducted in nor-
mobaric normoxia.
MATERIALS AND METHODS
Subjects
Thirteen male athletes (nine triathletes, two cross-
country skiers and two cyclists) gave written consent
to participate in this study, which was approved by the
Australian Institute of Sport Ethics Committee. Sub-
jects were ranked according to the power output
achieved during the last 2 min of an incremental cycle
ergometer test, that also established their peak oxygen
consumption ( _
V
O
2peak
). The ranking was used to
assign subjects to two ®tness-matched groups: the
CON group (n7) and LHTL group (n6). The
physical characteristics of the CON and LHTL groups
and their training frequency, intensity and duration did
not differ (Table 1). The nine triathletes (four CON
and ®ve LHTL) trained together and the remaining
athletes completed their own sport-speci®c training
schedules.
Experimental design
The study was conducted in Canberra, Australia at 600
m altitude, P
B
711 mmHg. The LHTL subjects spent
9.5 h night
±1
for 23 consecutive nights in a room
where enriched nitrogen produced hypoxia that simu-
lated 3000 m altitude (normobaric hypoxia;
O
2
15.48%). The CON subjects slept in their own
homes under normobaric normoxia. Training and
daytime living for all subjects was at an altitude of
600 m.
Submaximal workloads. After one habituation trial,
subjects completed ®ve, four-stage submaximal cycle
ergometer tests before, during and after the LHTL
group slept at simulated altitude. The timing of these
submaximal ergometer tests was 4 and 5 days before
(PRE), 2 and 3 days after (POST), as well as after 11 of
the 23 nights of simulated altitude (MID) (Fig. 1). All
tests were completed under normobaric normoxic
conditions in Canberra on one ergometer (Excalibur
Sport model, Lode, Groningen, Holland) that was
dynamically calibrated with a torquemeter. Based on the
habituation trial, workloads for each subject were pro-
grammed using the Lode `hyperbolic' mode at 1.5, 2.5,
3.5 and 4.5 W kg
±1
; and for the baseline test (day
5-PRE) these corresponded to an overall group mean
of 36, 52, 68, and 84% _
V
O
2peak
. The workloads pro-
grammed on day 5-PRE were replicated for each sub-
ject's subsequent tests and their cadence on day 5-PRE
was recorded each minute and then matched for the
four subsequent tests. Controlling cadence was a
necessary precaution when using the `hyperbolic' mode
of the Lode ergometer because in this condition power
output is constant and cadence independent, and yet
cadence can markedly alter the _
V
O
2
of cycling
(Woolford et al. 1999).
All-out trials. On days 5-PRE, 11-MID and 2-POST
an `all-out' trial was conducted in which the submaxi-
mal ergometer test was followed by 4 min of rest and
then by a 4-min maximal effort. The ®rst 2 min was set
at an individual load (mean  SD 5.6  0.4 W kg
±1
)
equivalent to 105% of the workload achieved at
_
V
O
2peak
in the habituation incremental test, and the last
2 min was an `all-out' effort. The 2 min workload at
5.6  0.4 W kg
±1
was programmed using the `hyper-
bolic' mode of the Lode ergometer, after which the
2 min all-out workload reverted immediately to the
`linear' mode of the ergometer with the linear factor
(gearing) programmed according to individual require-
ments. In the `linear' mode, power output on the Lode
is cadence dependent and appropriate gearing is
important for optimal performance. For each subject,
the hyperbolic and linear factors used during the
Table 1 Physical and training characteristics. The live high:train low
group (LHTL, n= 6) lived at 3000 m simulated altitude and trained at
600 m (Canberra, Australia), while the control group (CON, n=7)
lived and trained in Canberra. The data for peak oxygen consumption
(_
V
O
2peak
) and `all-out' 2-min power output are those achieved during
habituation (see Fig. 1). Data are mean and (SD). No signi®cant
differences were found between groups for any variable
Variable LHTL CON
Age (year) 25.4 (3.6) 25.1 (5.2)
Height (cm) 183.5 (10.0) 181.2 (6.3)
Body mass (kg) 73.0 (6.7) 73.3 (6.1)
_
V
O
2peak
(L min
±1
) 5.08 (0.34) 4.95 (0.45)
All-out 2-min power output (W kg
±1
) 5.74 (0.46) 5.72 (0.31)
Training (sessions week
±1
) 7.1 (2.3) 6.8 (2.3)
Training intensity (Borg units) 13.8 (1.1) 13.6 (1.0)
Training (h week
±1
) 13.4 (3.8) 10.6 (5.7)
276 Ó2001 Scandinavian Physiological Society
Hypoxia increases ef®ciency and muscle buffering C J Gore et al. Acta Physiol Scand 2001, 173, 275±286

habitation trial were replicated for the three subsequent
all-out tests. During the all-out effort, total work, _
V
O
2
and _
V
O
2peak
were recorded.
Biopsy trials. On days 4-PRE and 3-POST a `biopsy
trial' was conducted in which each subject had two
muscle biopsies (vastus lateralis), one at rest 30 min
before the four-stage submaximal ergometer test and
a second biopsy taken immediately (<15 s) after
completing 2 min at 5.6  0.4 W kg
±1
(Fig. 1).
Subject preparation and analyser calibration
Simulated altitude. Throughout each of the 23 nights,
%O
2
and %CO
2
inside the hypoxic room were meas-
ured every 30 min with Ametek (Pittsburgh, PA, USA)
O
2
and CO
2
gas analysers (model S-3A and CD-3A,
respectively) calibrated every 2 h at two points; with air
from outside the laboratory and with one precision
grade gas (BOC Gases Australia, Sydney, Australia).
The LHTL subjects had their resting heart rate (HR)
and blood oxyhaemoglobin saturation (S
p
O
2
) estimated
with ®nger-tip pulse oximetry (model 505-US, Criticare,
Waukesha, WI, USA) every 30 min.
Morning resting blood acid-base status. Resting venous
blood was collected under normoxic conditions within
30 min of waking for both LHTL and CON. Samples
were taken on the sixth day before entering the altitude
house (6-PRE); after 3, 5, 12 and 22 nights at simulated
altitude (A3, A5, A12 and A22, respectively), as well as
after one night of sleeping in normoxia (2-POST) for
determination of acid±base variables (Fig. 1). With each
subject supine, blood was sampled from a super®cial
forearm vein via a winged infusion set into a hepari-
nized 2 mL blood gas syringe. Resting samples were
also analysed for red blood cell parameters, with data
reported elsewhere (Ashenden et al. 1999).
Exercise blood sampling. Before each of the ®ve cycle
ergometer tests, a catheter was inserted into a super-
®cial dorsal hand vein and covered with an adhesive
plastic dressing and latex glove. After catheterization,
each subject was seated on the cycle ergometer and the
catheterized hand was immersed in a water bath
(44.5 °C) to ensure arterialization of venous blood.
After 10 min in this posture, a 1.5-mL pre-exercise
blood sample was acquired via a heparinized 2 mL
blood gas syringe. Blood samples (1.5 mL) were taken
from a dorsal hand vein during the last 30 s of each of
the four submaximal workloads and at
5.6  0.4 W kg
±1
during the biopsy trials, and on the
days of the all-out trial during the last 15 s of the ®nal
2-min effort.
Blood analyses. Blood samples were stored on ice
(<1 h) until analysis in triplicate for plasma pH and
bicarbonate concentration [HCO
3±
], lactate concentra-
tion [La
±
]
p
, and carbon dioxide partial pressure (P
CO
2
)
using an automated analyser (ABL System 625,
Figure 1 Testing schedule and simulated altitude exposure of control (CON, n7) and live high:train low (LHTL, n6) groups. Both the
CON and LHTL trained in normobaric normoxia in Canberra (600 m altitude), Australia, while LHTL spent 23 nights in normobaric hypoxia.
Ó2001 Scandinavian Physiological Society 277
Acta Physiol Scand 2001, 173, 275±286 C J Gore et al. Hypoxia increases ef®ciency and muscle buffering

Radiometer, Copenhagen, Denmark), which was calib-
rated daily in accordance with the manufacturer's
speci®cations.
Muscle biopsies and analyses
A needle biopsy sample was taken at rest from the
vastus lateralis muscle via one of the two incisions
made ipsilaterally under local anaesthesia (Xylocaine,
1%; Astra Pharmaceuticals, Sydney, Australia), with
suction applied to the needle. Both biopsies in a trial
were taken from separate incisions in the same leg, with
the exercise sample taken from an incision 1.5 cm
distal to the rest sample. All biopsies were taken at
constant depth by the same, experienced medical
practitioner. The second sample was taken immediately
after cessation of the 2 min exercise trial at
5.6  0.4 W kg
±1
, with the subject lying supported on
the cycle ergometer. The samples for metabolite and
bm analyses were rapidly frozen in liquid nitrogen.
Muscle pH, buffer capacity and total protein content. Before
analysis in duplicate, the samples were freeze-dried
(Modulo, Edwards, Crawley, UK) and dissected free of
connective tissue, blood and fat. The sample was
diluted 1:200 in 5 m
M
NaIAA, 145 m
M
KCl, and
10 m
M
NaCl, pH 7.0 and then homogenized (Omni
1000, Omni International, Warrenton, VA, USA) on ice
for 60 s. Muscle homogenate pH (expressed as [H
+
])
was measured at 37 °C under magnetic stirring with a
glass microelectrode (MI-145, Microelectrodes, Bed-
ford, TX, USA). The in-vitro buffer capacity (bm) was
then measured by titration of the homogenate from
pH 7.1 to 6.1 and expressed relative to muscle dry mass
(lmol H
+
g muscle dm
±1
pH
±1
). Total protein content
was determined spectrophotometrically (Lowry et al.
1951). The reliability of the duplicate measures was
calculated as the within subject standard deviation or
typical error of measurement (TEM) (Hopkins 2000).
The TEM for bm was 3.6 lmol H
+
g muscle dm
±1
pH
±1
or 1.9% of the mean, and the corresponding
values for total protein were 0.016 mg (mg muscle)
±1
equivalent to 1.2% of the mean.
Muscle metabolites. The muscle lactate (La
±
m
), adeno-
sine triphosphate (ATP), PCr, creatine (Cr) and
glycogen contents were measured in triplicate on freeze-
dried muscle using standard ¯uorometric techniques
(Lowry & Passoneau 1972). Muscle ATP, PCr and Cr
contents were corrected to the total Cr content. Muscle
anaerobic ATP production was estimated from the rest
to end-exercise changes (D) in ATP, PCr and La
±
m
, and
calculated as DATP + DPCr + 1.5DLa
±
m
. Because of
technical dif®culties, the sample size for these measures
was n4 and n6 for the LHTL and CON,
respectively. The respective TEMs for La
±
m
, ATP, PCr
and glycogen were 1.5, 0.6, 1.5 and 14 mmol kg dm
±1
equivalent to 2.0, 2.5, 5.8 and 2.5% of the mean values.
Oxygen consumption and mechanical ef®ciency
During each cycle ergometer test _
V
O
2
, carbon dioxide
output ( _
V
CO
2
), minute ventilation ( _
V
E
) and respiratory
exchange ratio (RER) were measured continuously and
results were displayed every 30 s. Data from the last 60 s
of each of the four 4-min submaximal workloads were
used to indicate the `steady-state' level, and _
V
O
2peak
was
determined as the highest value recorded in any 60-s
interval during the last 4 min of the all-out trial. The
open-circuit indirect calorimetry system comprised
Ametek O
2
and CO
2
gas analysers as well as two chain-
compensated gasometers and has been described pre-
viously (Pierce et al. 1999). The analysers were calibrated
before, and checked for drift after, each test using three
agrade gases (BOC Gases Australia). The average TEM
for _
V
O
2
was 0.12 and 0.09 L min
±1
, respectively, for
the duplicated PRE (5- and 4-PRE) and POST (2- and
3-POST) four stages of submaximal ergometry. At any
submaximal workload the mean difference between
either of the two repeat tests was <52 mL min
±1
for
both CON and LHTL groups. The corresponding PRE
and POST TEMs for _
V
E
during submaximal ergometry
were 5.0 and 4.7 L min
±1
, equivalent to 6.1 and 4.9% of
the respective mean values.
Gross mechanical ef®ciency (%) was determined
from the ratio of power output (kJ min
±1
) to energy
expended (kJ min
±1
), as calculated from _
V
O
2
and RER
(Elia & Livesey 1992).
Heart rate
Overnight resting heart rate (HR) each night was cal-
culated for the LHTL group as the grand mean from
11:00
PM
to 05:00
AM
. The HR during cycle ergometry
was assessed every 5 s by telemetry (Polar Vantage,
Polar Electro OY, Kempele, Finland). The TEMs for
HR during the four-stage submaximal ergometer tests
at PRE and POST were 3 and 4 beats min
±1
, equivalent
to 2.6 and 3.5% of the respective mean values.
Statistical analysis
All values are reported as mean  SD. The physical
and training characteristics of the two groups were
assessed with independent t-tests. Three-way analysis of
variance (
ANOVA
) with repeated measures was used to
test for interaction and main effects for most of the
dependent variables measured during exercise. The
three factors were group (CON and LHTL), day (PRE,
MID and POST simulated altitude), and stage of
278 Ó2001 Scandinavian Physiological Society
Hypoxia increases ef®ciency and muscle buffering C J Gore et al. Acta Physiol Scand 2001, 173, 275±286

exercise (rest, end of exercise and where relevant the
four submaximal workloads). When the three-way
interaction was not signi®cant, the data of LHTL and
CON groups were analysed with separate two-way
repeated measures
ANOVA
for day and stage of exercise.
Peak exercise data were analysed with two-way repeated
measures
ANOVA
for group by day. When interactions
or main effects achieved statistical signi®cance, Tukey
post hoc tests were used to identify differences between
cell means. Statistical signi®cance was tested at the
P< 0.05 level using Statistica software (StatSoft, Tulsa,
OK, USA). In addition, and as a method to partially
circumvent the likelihood of a type II error as a con-
sequence of our small sample size, the effect size
[ES (mean
1
± mean
2
)/SD] was calculated for selec-
ted results that did not achieve signi®cance and the
pooled SD was calculated when the SDs were unequal
(Cohen 1988). Cohen's (Cohen 1988) conventions for
effect size were adopted for interpretation, where
ES 0.2, 0.5 and 0.8 are considered as small, medium
and large, respectively.
RESULTS
All-out trials
Performance and _
V
O
2
.The _
V
O
2peak
of LHTL fell
signi®cantly by ±3.8  1.9% at MID and ±7.2 
4.1% at POST, whilst CON _
V
O
2peak
was unchanged
(Table 2). Total V
O
2
in the 2 min all-out effort was
also signi®cantly depressed in LHTL at POST com-
pared with PRE, although, the corresponding work
output was not changed in either group (Table 2).
Total V
O
2
during 2 min at 5.6  0.4 W kg
±1
was not
different between groups (P> 0.2) but tended to be
less after 23 nights of sleeping in hypoxia in LHTL
(DPRE vs. POST ±4.0%) than in CON
(D1.1%).
Cadence at PRE ranged from 90  7 to
102  2 rev min
±1
for 1.5 W kg
±1
and all-out work-
loads, respectively, for LHTL, and 93  15 to 102 
3 rev min
±1
for CON. No signi®cant differences were
found between groups or between different days of
exercise.
Submaximal _
V
O
2
and mechanical ef®ciency. During the
®rst four stages of the all-out trial, LHTL had a signi-
®cantly lower submaximal _
V
O
2
at both MID
()3.1  2.9%) and POST (±4.4  3.3%) compared
with PRE (Fig. 2). Submaximal _
V
E
was signi®cantly
increased after 23 nights of sleeping in moderate hyp-
oxia (Fig. 2). Although RER of LHTL was not signi-
®cantly different between days, the effect sizes tended
to be large (at 1.5 W kg
±1
PRE vs. MID, ES 2.27;
PRE vs. POST, ES 1.67). Overall, RER for LHTL
was 0.88  0.07 PRE and 0.91  0.07 POST. The
CON showed no change in submaximal _
V
O
2
,_
V
E
and
RER for MID and POST vs. PRE (Fig. 2).
Submaximal ef®ciency of LHTL was signi®cantly
different between days and stage of exercise (P0.02).
Each POST value (16.6  1.5, 19.6  0.8, 20.9  0.7
and 21.5  0.7%) was higher than the corresponding
PRE value (15.8  1.4, 18.7  0.9, 20.2  1.0 and
21.0  0.7%) at 1.5, 2.5, 3.5 and 4.5 W kg
±1
, respect-
ively. Overall, submaximal ef®ciency of the LHTL
group was improved 0.8% from PRE (18.9  2.7%) to
POST (19.7  2.4%) (P< 0.01).
Heart rate. Submaximal HR was signi®cantly differ-
ent between groups when comparing the three test days
and four submaximal stages of exercise [F
(6,66)
2.43,
P0.03). The PRE HR was not different between
Table 2 All-out trials. Peak and total
_
V
O
2
, work, peak HR, and end exer-
cise [La
±
]
p
and pH for 2-min all-out
cycle ergometry. The groups and the
intervention are described in Table 1
and the timing of tests is illustrated in
Fig. 1. Data are mean and (SD)
Day of measurement
Variable Group Day 5-PRE Day 11-MID Day 2-POST
_
V
O
2peak
(L min
±1
) LHTL 5.08 (0.34) 4.90 (0.33)* 4.78 (0.36)*
CON 4.95 (0.45) 4.92 (0.47) 4.87 (0.44)
V
O
2total
in 2 min (L) LHTL 9.99 (0.72) 9.63 (0.71) 9.24 (0.66)*
CON 9.60 (1.09) 9.77 (0.93) 9.64 (0.92)
Work in 2 min (kJ) LHTL 50.0 (4.2) 51.0 (3.9) 49.2 (4.2)
CON 50.5 (6.0) 51.5 (6.5) 50.3 (5.8)
Heart rate
peak
(beats min
±1
) LHTL 183 (9) 185 (6) 183 (6)
CON 189 (8) 189 (9) 190 (9)
[La
±
]
p
(mmol L
±1
) LHTL 15.4 (3.3) 16.7 (2.5)17.3 (2.6)
CON 17.4 (1.2) 21.1 (3.1)* 22.4 (1.7)*
pH LHTL 7.26 (0.03) 7.25 (0.03) 7.26 (0.03)
CON 7.24 (0.03) 7.23 (0.03) 7.24 (0.02)
*Signi®cantly different from PRE.
Signi®cantly different between groups on the same day.
Ó2001 Scandinavian Physiological Society 279
Acta Physiol Scand 2001, 173, 275±286 C J Gore et al. Hypoxia increases ef®ciency and muscle buffering

groups at any workload, however, HR was signi®cantly
lower for LHTL than CON at the ®rst three submax-
imal workloads at both MID and POST (Fig. 2). In
addition, HR of LHTL during the ®rst two stages of the
MID test and the ®rst stage of the POST test were
signi®cantly lower (6±8 beats min
±1
) than at PRE. The
HR
peak
was not different within or between groups for
PRE vs. POST (Table 2).
Blood biochemistry. The three-way interaction between
groups, test days and the ®ve stages of rest or sub-
maximal exercise was signi®cant for [La
±
]
p
Figure 2 Oxygen consumption ( _
V
O
2
), ventilation ( _
V
E
), respiratory
exchange ratio (RER) and heart rate (HR) for Live High:Train Low
(LHTL, n6, left panels) and Control (CON, n7, right panels)
groups during submaximal cycle ergometry before (PRE), after 11
nights (MID), and 2 days after (POST) 23 nights of simulated altitude.
Values are mean and SD. Signi®cant differences within groups; *MID
vs. PRE, POST vs. PRE; signi®cant differences between groups at
matched time, §MID vs. MID, àPOST vs. POST. Main effects for
Day (PRE, MID, POST), exercise stage (1.5±4.5 W kg
±1
), as well as
the day by stage interaction are indicated in each subpanel.
Figure 3 Arterialized venous plasma lactate concentration [La
±
]
p
,
CO
2
tension (P
CO
2
), pH and bicarbonate ion concentration [HCO
3
±
]
for the LHTL (left panel) and CON (right panel) groups as described in
Fig. 2. Values are mean and SD. Signi®cant differences within group;
*MID vs. PRE, POST vs. PRE; signi®cant differences between
groups at matched time, §MID vs. MID, àPOST vs. POST.
280 Ó2001 Scandinavian Physiological Society
Hypoxia increases ef®ciency and muscle buffering C J Gore et al. Acta Physiol Scand 2001, 173, 275±286

(F
(8,88)
2.22, P0.03). During submaximal exercise,
[La
±
]
p
for LHTL was not different between test days,
but within CON [La
±
]
p
at 4.5 W kg
±1
was signi®cantly
higher than PRE at both MID and POST (Fig. 3).
Furthermore, [La
±
]
p
for LHTL was signi®cantly lower
than that of CON at MID at 3.5 W kg
±1
, and at both
MID and POST at 4.5 W kg
±1
. At the end of the 2 min
all-out effort, LHTL [La
±
]
p
was not different between
PRE, MID and POST tests, although CON [La
±
]
p
was
signi®cantly higher at both MID and POST than PRE
(Table 2).
The LHTL P
CO
2
during MID was lower than at
PRE at both 3.5 and 4.5 W kg
±1
, and at POST was
lower than PRE at 4.5 W kg
±1
(Fig. 3). Although Tukey
post hoc tests did not identify differences between cell
means, pH at 4.5 W kg
±1
tended to be higher compared
with PRE at both MID (ES 0.50) and POST
(ES 0.49). While not signi®cantly different between
days, LHTL [HCO
3
±
] at rest and during submaximal
exercise tended to be lower at both MID and POST
compared with PRE (ES 0.5). At the end of all-out
exercise, pH was stable in each group for the three tests
(Table 2).
Biopsy trials
Performance and _
V
O
2
.The LODE ergometer was
programmed to ensure that the amount of work com-
pleted during 2 min at 5.6  0.4 W kg
±1
was the same
on both days (48.2  5.1 kJ). There was a non-signi®-
cant (P> 0.20) trend for total V
O
2
during the 2 min to
be lower in LHTL (D±3.5%) but not CON
(D0.2%).
Muscle buffer capacity and metabolites. Resting bm
increased signi®cantly in LHTL (17.7  4.9%) but was
unchanged in CON (0.5  5.8%, Fig. 4). Analysis of bm
in post-exercise samples con®rmed this ®nding of an
elevation in the LHTL group only (data not shown). The
increased bm was not the result of an increased total
muscle protein content, as the latter did not differ
between or within groups (Table 3). Muscle [H
+
] was not
signi®cantly different between the groups. The pooled
data of both groups indicated no difference in resting
muscle [H
+
] PRE vs. POST (70.0  4.1 vs.
66.9  4.9 nmol kg dm
±1
, respectively), although at the
end of exercise it tended to be lower POST than PRE
[140.2  19.6 vs. 159.7  21.6 nmol kg dm
±1
; day by
exercise interaction (P0.06)]. The [H
+
] accumulation
and calculated in vivo bm after exercise at
5.6  0.4 W kg
±1
(D[H
+
]) was unchanged in either
LHTL or CON (Table 3). Muscle ATP, PCr and glyco-
gen decreased with exercise whereas Cr increased, but
these were not different between groups nor affected by
23 nights sleeping in hypoxia (Table 3). The La
±
m
accu-
mulation (DLa
±
m
) and estimated anaerobic energy pro-
duction after exercise at 5.6  0.4 W kg
±1
was
unchanged from PRE to POST in either group (Table 3).
Heart rate. No differences between or within groups
were found for PRE vs. POST HR at 5.6  0.4 W kg
±1
(Table 4).
Blood biochemistry. At 5.6  0.4 W kg
±1
[La
±
]
p
,P
CO
2
,
pH, and [HCO
3
±
] were not different between groups or
from PRE to POST (Table 4).
Morning blood biochemistry
Morning resting plasma pH was not different between
groups at baseline or after one night of sleeping in
hypoxia, but was signi®cantly higher at day A5 in LHTL
than in CON (Fig. 5). Within LHTL, pH at A5 tended
to be higher than at baseline (P0.07, ES 2.29).
Morning resting [HCO
3
±
] was not different between
groups on any day, although it tended to be lower
(at least 1.2 mmol L
±1
) in LHTL during and 2 days
after simulated altitude (Fig. 5). The between group
effect sizes at days A3 and A22 were 0.51 and 1.04,
respectively.
Overnight heart rate and blood saturation
Overnight resting HR of the LHTL group was
unchanged across the 23 nights, with a grand mean of
57  11 beats min
±1
, and the S
p
O
2
was 91  3% for
the 219 h spent in normobaric hypoxia.
DISCUSSION
Our major ®ndings challenge conventional concepts
of adaptation to chronic hypoxic exposure. We show
m
Figure 4 Change in resting in-vitro muscle buffering capacity (bm)
PRE and POST 23 nights of simulated altitude. Left panel shows
individual data points of LHTL group (n6) that lived high and
trained low with mean  SD data indicated with large symbols. The
right panel is for the control (CON, n7) group.
Ó2001 Scandinavian Physiological Society 281
Acta Physiol Scand 2001, 173, 275±286 C J Gore et al. Hypoxia increases ef®ciency and muscle buffering

for the ®rst time that muscle in-vitro buffer capacity
was increased after sleeping in hypoxia, and thus can
be attributed to chronic hypoxic exposure alone.
However, after LHTL this did not coincide with
enhanced muscle H
+
regulation, evidenced by an
unchanged post-exercise muscle [H
+
], or by a general
up-regulation of anaerobic metabolism during intense
exercise. The second major ®nding was that whole
body _
V
O
2
during submaximal cycle ergometry under
normobaric, normoxic conditions was signi®cantly
lower after 23 nights of sleeping at 3000 m simulated
altitude. The ®nding of reduced _
V
O
2
at a constant
exercise workload, without a corresponding elevation
in anaerobic metabolism suggests that 23 nights
exposure to moderate hypoxia enhances mechanical
ef®ciency during exercise.
Muscle buffer capacity, anaerobic metabolism
and acid±base regulation
This is the ®rst study to report that merely sleeping,
rather than living and training, in hypoxia elevates bm
and thus strongly suggests that hypoxia is the key factor
in improving bm. The increase in bm was not the result
of increased muscle protein content but apparently
re¯ected a qualitative change in the buffer capacity of
the dipeptides or protein expressed. This may be a
consequence of a higher intramuscular carnosine con-
centration as suggested by others (Saltin et al. 1995a),
but the mechanism remains unknown. Elevated bm
after LHTL is consistent with the 5±6% increase
reported after training and living at 2000±2700 m
(Mizuno et al. 1990, Saltin et al. 1995a). In contrast, a
recent report indicated an unspeci®ed decrease in bm
after living at 2500 m and training at 2200±3000 m
(Stray-Gundersen et al. 1999).
This is also the ®rst report of the effects of LHTL on
skeletal muscle H
+
regulation during exercise. Surpris-
ingly, there did not appear to be a positive modulation
of intramuscular H
+
regulation, with an unchanged
post-exercise muscle [H
+
] during intense exercise after
LHTL, in comparison with the CON group. Such an
effect with LHTL would be expected to be evident from
Table 3 Biopsy trials. Muscle protein content, H
+
concentration and metabolites at rest and immediately after 2 min of cycle ergometry at
5.6  0.4 W kg
±1
. The groups and the intervention are described in Table 1 and the timing of tests is illustrated in Fig. 1. The sample size for
ATP, PCr, glycogen, Cr and La
±
m
are n= 4 and n= 6 for the LHTL and CON groups, respectively. Data are mean and (SD). Differences within
and between groups are not signi®cant
LHTL CON
Variable Condition Day 4-PRE Day 3-POST Day 4-PRE Day 3-POST
Protein (mg (mg muscle
±1
)) Rest 0.177 (0.010) 0.174 (0.013) 0.168 (0.014) 0.166 (0.015)
Exercise 0.175 (0.013) 0.174 (0.012) 0.168 (0.012) 0.166 (0.014)
D(Ex ± Rest) 38.6 (6.6) 34.0 (12.7) 42.6 (2.4) 43.4 (3.4)
[H
+
] (nmol L
±1
) Rest 71.1 (3.8) 67.3 (5.5) 69.0 (4.4) 66.4 (4.8)
Exercise 156.6 (22.8) 139.9 (20.4) 162.3 (21.9) 140.5 (20.5)
D(Ex ± Rest) 85.5 (21.7) 72.6 (19.2) 93.3 (18.3) 74.1 (22.5)
b
in-vivo
=(D[H
+
]/D[La
±
]
m
) 105.6 (30.0) 104.7 (40.1) 115.4 (17.8) 136.5 (30.0)
ATP Rest 28.6 (0.8) 28.6 (0.6) 28.7 (1.2) 28.7 (1.0)
(mmol kg dm
±1
) Exercise 18.0 (0.4) 18.1 (0.5) 18.0 (0.3) 17.9 (0.4)
PCr Rest 87.9 (0.7) 87.9 (0.5) 87.9 (3.0) 89.0 (1.9)
(mmol kg dm
±1
) Exercise 62.5 (0.9) 62.3 (0.7) 62.9 (0.7) 63.0 (1.1)
Glycogen Rest 576 (98) 571 (79) 611 (68) 599 (56)
(mmol glucosyl units kg dm
±1
) Exercise 245 (15) 247 (10) 235 (19) 227 (27)
Cr Rest 47.5 (1.4) 47.6 (1.2) 47.7 (1.3) 47.7 (1.3)
(mmol kg dm
±1
) Exercise 72.9 (1.0) 73.1 (1.1) 73.8 (0.7) 73.7 (0.9)
[La
±
]
m
Rest 5.9 (2.9) 6.0 (2.5) 4.9 (0.6) 4.9 (0.5)
(mmol kg dm
±1
) Exercise 44.5 (7.1) 40.1 (12.1) 47.5 (2.5) 48.2 (3.4)
Anaerobic ATP production D(Rest to end Ex) 93.9 (9.7) 87.1 (18.9) 100.7 (3.7) 101.9 (5.8)
(mmol kg dm
±1
)
Table 4 Biopsy trials. Plasma metabolites and acid±base status, as
well as HR at the immediate end of 2 min of cycle ergometry at
5.6  0.4 W kg
±1
. The groups and the intervention are described in
Table 1 and the timing of tests is illustrated in Fig. 1. Data are mean
and (SD). Differences within and between groups are not signi®cant
Variable Group Day 4-PRE Day 3-POST
[La
±
]
p
(mmol L
±1
) LHTL 9.3 (2.1) 11.3 (3.2)
CON 10.1 (2.3) 14.3 (4.0)
P
CO
2
(mmHg) LHTL 31.6 (1.9) 34.3 (2.5)
CON 32.5 (2.1) 33.8 (2.4)
pH LHTL 7.34 (0.03) 7.35 (0.04)
CON 7.34 (0.02) 7.34 (0.03)
HCO
3
±
(mmol L
±1
) LHTL 14.5 (1.8) 14.2 (1.7)
CON 15.1 (1.5) 14.4 (1.5)
Heart rate (beats min
±1
) LHTL 166 (9) 168 (6)
CON 169 (9) 172 (9)
282 Ó2001 Scandinavian Physiological Society
Hypoxia increases ef®ciency and muscle buffering C J Gore et al. Acta Physiol Scand 2001, 173, 275±286

the matched work bout used in this study because
identical, rather than exhausting, work bouts are a sali-
ent method to compare markers of muscle metabolism
and ion regulation (Harmer et al. 2000). Further, the
calculated in-vivo bm was not enhanced after LHTL,
although considerable variability was found in the data.
This suggests that intramuscular H
+
regulation was not
improved after LHTL. As we measured [H
+
] in dried
muscle and CO
2
is lost during the freeze-drying process,
our [H
+
] values are slightly lower than expected in wet
muscle. Nonetheless, both F
E
CO
2
(data not shown) and
arterialized venous P
CO
2
were lower during exercise
after LHTL, suggesting that intramuscular CO
2
would
also tend be less in the LHTL group. Hence, the CO
2
-
dependent H
+
accumulation would be lower in LHTL,
consistent with our conclusions that an increased
bm was not associated with improved muscle H
+
regulation. Thus, our results are incompatible with the
concept that the primary importance of increased bmis
to confer bene®ts for muscle H
+
regulation. In addition
to bm, muscle H
+
regulation during exercise will be
affected by the sarcolemmal lactate
±
/H
+
and Na
+
/H
+
exchange mechanisms, capillarization and muscle blood
¯ow (Juel 1998) and by changes in the intracellular
strong ion difference (Kowalchuk et al. 1988). The
effects of LHTL on each of these remain unknown. An
interesting ®nding was that the increased bm in the
LHTL group occurred without any corresponding ele-
vation in other markers of anaerobic metabolism, in
contrast with the suggestion of others who used natural
altitude exposure (Mizuno et al. 1990, Saltin et al.
1995a); although with a small sample size our analyses
are prone to type II errors. The degradation of muscle
ATP, PCr and glycogen during intense exercise were
unchanged, as were the intramuscular and blood accu-
mulation of La
±
and H
+
ions. Each of these changes
was highly reproducible with low TEM, and was iden-
tical in the PRE and POST trials in both the CON and
LHTL groups. The decline in ATP was most likely
because of the 2-min exercise bout at 105% _
V
O
2peak
.
The work completed in this trial (48 kJ) was similar to
that in the last 2 min of the all-out trial (50 kJ), when
subjects were asked to produce as much work as
possible. Thus, reductions in ATP are not unexpected
with this heavy exercise. The decline in PCr and rise in
Cr was surprisingly small relative to the rise in La
±
. This
may re¯ect the usual slight delay in biopsy sampling and
a likely rapid PCr resynthesis in these endurance-trained
athletes. The anaerobic ATP production may conse-
quently be slightly underestimated, but importantly, this
was clearly not enhanced after LHTL.
The typical lactate response to exercise during
chronic altitude exposure is an initial elevation in lactate
accumulation in arterial and venous blood as well as in
muscle, together with elevated muscle lactate release,
each of these subsequently decline with acclimatization
(Hochachka 1988, Brooks et al. 1992, 1998, Reeves et al.
1992). Our data clearly demonstrate that La
±
accumu-
lation was not elevated during intense exercise after
LHTL. The muscle and blood lactate data are incon-
sistent with the premise that lactate production was
greater after sleeping in hypoxia, consistent with our
conclusion that anaerobic metabolism is not enhanced
after LHTL. The [La
±
]
p
also was not increased within
the LHTL group after 23 nights spent in hypoxia. The
[La
±
]
p
during the latter stages of exercise was lower in
LHTL than in CON subsequent to simulated altitude
but this was because of an unexpected increase in the
CON group. Thus, our data suggest it is unlikely that
the typical lactate response to natural altitude occurred
after LHTL, possibly because of both the simulated
altitude and duration of exposure being insuf®cient to
elicit such a response.
Figure 5 Morning resting plasma pH (top panel) and bicarbonate
concentration (bottom panel) of live high:train low (LHTL, n6)
and control (CON, n7) groups before, during and after LHTL
spent 23 nights sleeping in hypoxia. Values are mean and SD.
*Signi®cant difference between groups.
Ó2001 Scandinavian Physiological Society 283
Acta Physiol Scand 2001, 173, 275±286 C J Gore et al. Hypoxia increases ef®ciency and muscle buffering

Reduced submaximal oxygen consumption and enhanced
ef®ciency
A clear ®nding in the current study was that under
normoxic conditions _
V
O
2
of the LHTL group was
depressed and ef®ciency was increased at each of the
four, 4-min submaximal workloads after both 11 and 23
nights of sleeping in hypoxia (Fig. 3). These results
challenge the conventional concept that, at sea level,
_
V
O
2
at any given submaximal power output remains
unchanged after returning from an altitude or simulated
altitude sojourn (Levine & Stray-Gundersen 1997, Piehl
Aulin et al. 1998). Most other studies have reported no
change in submaximal _
V
O
2
at sea level (Wolfel et al.
1991, Grassi et al. 1996, Levine & Stray-Gundersen
1997, Piehl Aulin et al. 1998). However, our data are
consistent with a recent report that _
V
O
2
was signi®-
cantly lower (8±10%) during prolonged submaximal
cycle ergometry subsequent to a 21-day climb (2160±
6194 m) (Green et al. 2000b). Collectively, our data and
those of subjects living and climbing at natural altitude
(Green et al. 2000b) suggest that one can attribute the
increase in mechanical ef®ciency to hypoxia per se
rather than hypobaria, cold or the effects of heavy
athletic training. Interestingly, our results are consistent
with those of several cross-sectional studies that have
reported higher exercise ef®ciency in altitude natives
compared with lowlanders (Hochachka et al. 1991,
Saltin et al. 1995b). We cannot be completely sure why
our ®nding differs from those of most others. How-
ever, our indirect calorimetry system has good precision
and we were very careful to maintain identical pedalling
cadences for a subject across all exercise trials. Failure
to control cadence may have confounded the work of
others (Sutton et al. 1988, Wolfel et al. 1991) as for
example, a cadence of 90 vs. 120 rev min
)1
lowers
submaximal _
V
O
2
by an average of 0.47 L min
±1
(Woolford et al. 1999).
The reduction in whole body _
V
O
2
during exercise
after LHTL can likely be explained by a shift from fat
to carbohydrate oxidation, rather than a shift from
oxidative to anaerobic metabolism. In our study,
submaximal RER was marginally higher (0.03) POST
than PRE, which is suf®cient to entirely explain the
0.8% improvement in ef®ciency of exercise after
LHTL. The higher RER is consistent with the sugges-
tion that at altitude increased carbohydrate and lactate
¯uxes re¯ect an overall shift towards carbohydrate
utilization which optimizes the available energy for a
given oxygen consumption (Brooks et al. 1998).
Preferential use of carbohydrate fuels rather than fats at
4300 m altitude has been shown at rest and during
submaximal exercise (Brooks et al. 1992, Roberts et al.
1996a,b). Our ®ndings of a lower _
V
O
2
at the same
absolute workload are consistent with the postulate that
altitude acclimatization improves coupling of ATP
demand and supply (Hochachka 1988).
Other mechanisms which might contribute to
increased exercise ef®ciency after LHTL include a
reduction of ATP consuming processes within skeletal
muscle as shown recently by down-regulation of Na
+
,
K
+
-ATPase after an altitude sojourn (Green et al.
2000a), and a reduction in _
V
O
2
of the respiratory
musculature. The latter is unlikely because exercise
ventilation was increased after LHTL and during heavy
cycle exercise the respiratory muscles consume a
signi®cant fraction of the pulmonary _
V
O
2
(Harms et al.
1997). Lastly, type I ®bres are energetically more ef®-
cient when cycling (Coyle et al. 1992), and both ®bre
recruitment and cycling _
V
O
2
are cadence-dependent
(Barstow et al. 1996, Woolford et al. 1999). However, it
seems doubtful that the LHTL group may have
increased their type I ®bre recruitment because all
®bres would be expected to be recruited during the
maximal work bouts, when _
V
O
2peak
was also
subnormal. Reduced submaximal cycling cadence also
cannot explain the subnormal _
V
O
2
as this was main-
tained constant for each subject in all tests.
_
V
O
2
peak and performance after simulated altitude. A novel
®nding in this study was that after LHTL _
V
O
2peak
was
depressed by 7%, although total work was unchanged,
during 2 min of all-out cycling. It is implausible that
this decrease in _
V
O
2peak
could be explained by
detraining of the LHTL group. Even studies of well-
trained athletes who completely cease training for
2±3 weeks have reported a decrease in _
V
O
2max
of only
2±7% (Houston et al. 1979, Coyle et al. 1984, Laforgia
et al. 1999). Before and during the period of LHTL,
most of the CON group trained with the LHTL group
and the former maintained a stable _
V
O
2peak
throughout
this study. It therefore seems unlikely that the LHTL
group which spent 13 h week
±1
in athletic preparation
at a mean intensity of 13.8 Borg units would have
detrained. Finally, our _
V
O
2
system has high precision,
which suggests that measurement error was not the
cause for the observed reduction in _
V
O
2
. In this
context it is notable that tests on LHTL and CON
subjects were interspersed. Although there is a wide-
spread paradigm that acclimatization to hypoxia
increases red cell mass and consequently _
V
O
2max
(Cerretelli & Hoppeler 1996, Rusko 1996, Levine &
Stray-Gundersen 1997, Rodrõ
Âguez et al. 1999, Fulco
et al. 2000), we (Hahn & Gore 2001) and others (Sawka
et al. 2000) oppose this view. Our LHTL subjects
exhibited no change in haemoglobin mass or reticulo-
cyte indices of accelerated erythropoiesis (Ashenden
et al. 1999) but their _
V
O
2peak
was depressed. The
potential mechanisms underlying decreased _
V
O
2peak
require further investigation.
284 Ó2001 Scandinavian Physiological Society
Hypoxia increases ef®ciency and muscle buffering C J Gore et al. Acta Physiol Scand 2001, 173, 275±286

Total work as an indicator of performance was
unchanged in the LHTL group but this may be a problem
of insuf®cient statistical power. There is mounting
evidence from three independent groups that LHTL may
yield small improvements (0.8±1.3%) in events lasting
from 50 s to 17 min; 400 m sprint (Nummela &Rusko
2000), 4-min all-out effort (Hahn et al. 2001), as well as
3000 m (Stray-Gundersen et al. 2001) and 5000 m
(Levine & Stray-Gundersen 1997) run times.
CONCLUSIONS
Chronic nightly hypoxic exposure using the LHTL
model for 23 days increased bmby18%, but this
occurred in the absence of enhanced muscle H
+
regulation during intense exercise. Living high:training
low also signi®cantly reduced whole body oxygen
utilization during exercise in normoxia, including
during standardized submaximal exercise workloads,
and by 7% at _
V
O
2peak
. Thus, submaximal cycling
ef®ciency was increased by 0.8%, which could be
attributed to increased carbohydrate oxidation. Our
results suggest that increased bm may be merely an
indicator of adaptation and that greater ef®ciency may
be of more practical importance.
This work is dedicated to the memory of James Ganley who assisted
with all data collection. The authors wish to thank K. Gawthorn,
H. Lee, J. Dixon, S. Angheli, K. Lynch, E. Lawton and R. Shugg for
technical assistance. We thank R. Parisotto and T. Boston for blood
analyses, R. Spence for engineering support with the Altitude House
and Associate Prof. M. Carey for assistance with the muscle
biochemical analyses. The technical and ®nancial support from
BOC Gases Australia is gratefully acknowledged as is the funding
provided by the Australian Sports Commission.
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