Hypoxic Training
Seventeen physically active male subjects (mean ± SD: age, 27 ± 3 yr; height, 181 ± 7 cm; body mass, 78 ± 9 kg; V̇O2peak, 47 ± 5 mL·kg·min) participated in this study. Body composition was assessed by a dual-energy x-ray absorptiometer scan (Lunar iDXA™ GE Healthcare, Madison, WI). This study involving human subjects was approved by the ethical committee for the Eidgenössische Technische Hochschule Zürich (EK 2011-N-51) in accordance with the Declaration of Helsinki. Before the start of the experiments, a written informed consent was obtained from all participants.
The experimental design consisted of i) familiarization period followed by 2 wk of lead-in training phase, ii) baseline testing, iii) experimental intervention, i.e., a 6-wk endurance training period in either normoxia or normobaric hypoxia, and iv) postintervention measurements.
Familiarization consisted of an incremental cycling test to exhaustion followed by a time trial. Each subject repeated the familiarization procedure on two separate occasions before undergoing the baseline measurements. During the 2-wk period preceding the baseline measurements, the subjects performed six sessions of low-volume high-intensity training. The objectives of this 2-wk period were i) to homogenize the physical activity of subjects before starting the intervention period (lead-in training) and ii) to separately investigate the effect of low-volume high-intensity training.
Incremental Exercise to Exhaustion. Subjects performed an incremental cycling test to volitional fatigue on an electronically braked cycle ergometer (Monark, Varberg, Sweden) to determine maximal workload (Wmax). Minute ventilation and O2 and CO2 concentrations in the expired gas were continuously measured by using an online breath-by-breath gas collection system (Innocor; Innovision, Odense, Denmark). The gas analyzers and the flowmeter of the applied spirometer were calibrated before each test. Subjects began the exercise at three consecutive 5-min workloads at a fixed pedaling cadency, increasing by 50-W increments, beginning with 50 W and finishing with 150 W. From that point, the workload was increased by 30 W every 75 s thereafter until volitional fatigue. Wmax was calculated as Wmax = Wcompl + 30(t/90), where Wcompl is the last completed workload and t is the number of seconds that the final, not completed, workload was sustained and 30 W is the workload increment. V̇O2peak was determined as the highest value averaged over 30 s for each subject. The subjects were then allowed a 1-h rest before beginning a time trial.
The reproducibility of our gas collection system was assessed during a separate experiment in seven subjects who performed duplicate incremental tests until exhaustion (on different days) in normoxia and in acute normobaric hypoxia. The coefficient of variation for V̇O2peak, expressed as percent typical error (i.e., SD of difference scores/√2), was 3.5% in normoxia and 3.6% in hypoxia. The present gas collection system was also tested against another metabolic cart (Quark; COSMED, Rome, Italy) during an incremental exercise to exhaustion in four subjects, with the two systems connected in series. In this setting, the percent typical error was 1.4%.
Time Trial. After rest, each subject performed a time trial in which they completed a given workload as quickly as possible. The criterion for completion of the time trial was completing a set relative workload determined as 25% more work than that completed during the incremental cycling test at baseline (thus, the absolute work done before and again after exercise was the same absolute workload). This relative workload across subjects was selected for the time trial exercise tests because, first, it controlled for differences in aerobic capacity across subjects and, second, it made all baseline tests last approximately 20 min (1246 ± 215 s). This allowed for a more standardized test of endurance across all subjects opposed to a test with a set absolute workload. All time trials were performed on an electronically braked ergometer. The ergometer was set to newton mode in an attempt to closely model more realistic race conditions so that each subject could increase or decrease resistance manually and/or via their cadence as desired to complete the predetermined workload. Subjects were instructed to complete the tests as quickly as possible and were provided no temporal or physiological feedback. The only feedback provided was the remaining workload until completion. Exercise duration and average power were recorded upon completion of each test.
Exercise Tests in Hypoxia. On a separate day, the subjects performed the same exercise procedure as in normoxia but in normobaric hypoxia (FIO2 = 0.15) (AltiTrainer; SMTEC, Nyon, Switzerland). Recovery between the two tests (incremental exercise then time trial) was performed in normoxia.
Subjects were paired according to their baseline V̇O2peak and randomly divided, in a double-blind, placebo-controlled manner, into two groups: eight subjects training in normoxia and the other nine in normobaric hypoxia (FIO2 = 0.15, corresponding to an altitude of approximately 2500 m). Subjects performed a total of 20 training sessions over 6 wk (3–4 sessions per week), each session lasting 60 min. Four different intensity profiles were alternately given to the subjects to keep motivation high (Table 1). In both groups (i.e., training in normoxia and training in hypoxia), subjects trained at the same relative workload, calculated from their individual Wmax previously determined in normoxia or hypoxia. To reach similar amounts of total work during training in both groups (because absolute workloads were lower during training in hypoxia), the subjects training in normoxia were furtively unloaded at the start and end of each session. During all training sessions, all subjects wore a face mask and inhaled into the air mixing system (AltiTrainer) connected to compressed air (delivering normoxia) or nitrogen (delivering hypoxia). By doing so, neither investigators nor subjects could identify the treatment. Subjects filled out a questionnaire after the training intervention, indicating that 47% guessed the right training group they were in.
Skeletal muscle biopsies were obtained from the vastus lateralis muscle at baseline and after the 6 wk of endurance training. Samples were collected under local anesthesia using the Bergström technique with a needle modified for suction. The biopsy was immediately dissected free of fat and connective tissue and divided into sections for measurements of mitochondrial respiration, as reported elsewhere. All biopsies were taken approximately 48 h after the last bout of exercise. Some of the samples were collected in the morning, and others were collected in the afternoon, but all biopsies were collected under the same standardized conditions. The procedures are also described in the present article (see Text, Supplemental Digital Content 1, http://links.lww.com/MSS/A440, which describes the procedures for mitochondrial respiration measurements).
Total hemoglobin mass (Hbmass) was measured with a carbon monoxide (CO) rebreathing technique, as previously described. Each time, the subject would come to the laboratory and first rest for 20 min in a semirecumbent position. Thereafter, 2 mL of blood was sampled from an antecubital vein via a 20-gauge catheter and analyzed immediately in quadruplicate for i) percent carboxyhemoglobin and Hb concentration ([Hb]) using a hemoximeter (ABL800; Radiometer, Copenhagen, Denmark); and ii) hematocrit (Hct) with the micromethod (4 min at 13,500 rpm). After baseline collection and control, the subject breathed 100% O2 for 4 min to flush the nitrogen from the airways. The breathing circuit (previously flushed with O2) was then closed, and a bolus (1.5 mL·kg) of 99.997% chemically pure CO (CO N47; Air Liquide, Paris, France) was administrated into the now closed rebreathing apparatus. The subject rebreathed this gas mixture for 10 min. At the end of the rebreathing period, an additional 2-mL blood sample was obtained and analyzed as before. The change in percent carboxyhemoglobin between the first and second measurement was used to calculate Hbmass, taking into account the amount of CO that remained in the rebreathing circuit at the end of the procedure (2.2%). Total RCV, blood volume, and plasma volume were derived from measures of Hbmass, [Hb], and Hct. All CO rebreathing tests were performed by the same researcher. Baseline and postendurance training values reported here correspond to the average of duplicate measurements conducted on separate days within these testing sessions. The coefficient of variation for Hbmass, assessed from duplicate measures and expressed as the percent typical error (i.e., SD of difference scores/√2), was 3.3% at baseline and 2.4% after endurance training. Of note, during baseline measurements, duplicate Hbmass recordings differed by 122 g in one subject. Without including this result, the typical error of Hbmass would have been 2.5% at baseline.
The Kolmogorov–Smirnov test was applied to examine the normality in the distribution of data. The Bartlett test was used to evaluate the uniformity of variance between conditions. Resting, maximal exercise, and endurance performance data were analyzed using a two-way ANOVA with repeated measures, which included one between-subject (group, i.e., normoxic training and hypoxic training) and one within-subject (timing, i.e., pretraining and posttraining) factor. When necessary, differences between values obtained pre- and posttraining for a particular group were analyzed using Student's paired t-test. The effect of acute hypoxia on exercise parameters for a particular group was analyzed using Student's paired t-test. Statistics were done with the StatView software, version 5.0. (SAS Institute, Cary, NC). The values are reported as arithmetic means ± SD. A P value of <0.05 was considered significant.
Methods
Subjects
Seventeen physically active male subjects (mean ± SD: age, 27 ± 3 yr; height, 181 ± 7 cm; body mass, 78 ± 9 kg; V̇O2peak, 47 ± 5 mL·kg·min) participated in this study. Body composition was assessed by a dual-energy x-ray absorptiometer scan (Lunar iDXA™ GE Healthcare, Madison, WI). This study involving human subjects was approved by the ethical committee for the Eidgenössische Technische Hochschule Zürich (EK 2011-N-51) in accordance with the Declaration of Helsinki. Before the start of the experiments, a written informed consent was obtained from all participants.
Experimental Design
The experimental design consisted of i) familiarization period followed by 2 wk of lead-in training phase, ii) baseline testing, iii) experimental intervention, i.e., a 6-wk endurance training period in either normoxia or normobaric hypoxia, and iv) postintervention measurements.
Familiarization and Lead-in Training
Familiarization consisted of an incremental cycling test to exhaustion followed by a time trial. Each subject repeated the familiarization procedure on two separate occasions before undergoing the baseline measurements. During the 2-wk period preceding the baseline measurements, the subjects performed six sessions of low-volume high-intensity training. The objectives of this 2-wk period were i) to homogenize the physical activity of subjects before starting the intervention period (lead-in training) and ii) to separately investigate the effect of low-volume high-intensity training.
Exercise Testing
Incremental Exercise to Exhaustion. Subjects performed an incremental cycling test to volitional fatigue on an electronically braked cycle ergometer (Monark, Varberg, Sweden) to determine maximal workload (Wmax). Minute ventilation and O2 and CO2 concentrations in the expired gas were continuously measured by using an online breath-by-breath gas collection system (Innocor; Innovision, Odense, Denmark). The gas analyzers and the flowmeter of the applied spirometer were calibrated before each test. Subjects began the exercise at three consecutive 5-min workloads at a fixed pedaling cadency, increasing by 50-W increments, beginning with 50 W and finishing with 150 W. From that point, the workload was increased by 30 W every 75 s thereafter until volitional fatigue. Wmax was calculated as Wmax = Wcompl + 30(t/90), where Wcompl is the last completed workload and t is the number of seconds that the final, not completed, workload was sustained and 30 W is the workload increment. V̇O2peak was determined as the highest value averaged over 30 s for each subject. The subjects were then allowed a 1-h rest before beginning a time trial.
The reproducibility of our gas collection system was assessed during a separate experiment in seven subjects who performed duplicate incremental tests until exhaustion (on different days) in normoxia and in acute normobaric hypoxia. The coefficient of variation for V̇O2peak, expressed as percent typical error (i.e., SD of difference scores/√2), was 3.5% in normoxia and 3.6% in hypoxia. The present gas collection system was also tested against another metabolic cart (Quark; COSMED, Rome, Italy) during an incremental exercise to exhaustion in four subjects, with the two systems connected in series. In this setting, the percent typical error was 1.4%.
Time Trial. After rest, each subject performed a time trial in which they completed a given workload as quickly as possible. The criterion for completion of the time trial was completing a set relative workload determined as 25% more work than that completed during the incremental cycling test at baseline (thus, the absolute work done before and again after exercise was the same absolute workload). This relative workload across subjects was selected for the time trial exercise tests because, first, it controlled for differences in aerobic capacity across subjects and, second, it made all baseline tests last approximately 20 min (1246 ± 215 s). This allowed for a more standardized test of endurance across all subjects opposed to a test with a set absolute workload. All time trials were performed on an electronically braked ergometer. The ergometer was set to newton mode in an attempt to closely model more realistic race conditions so that each subject could increase or decrease resistance manually and/or via their cadence as desired to complete the predetermined workload. Subjects were instructed to complete the tests as quickly as possible and were provided no temporal or physiological feedback. The only feedback provided was the remaining workload until completion. Exercise duration and average power were recorded upon completion of each test.
Exercise Tests in Hypoxia. On a separate day, the subjects performed the same exercise procedure as in normoxia but in normobaric hypoxia (FIO2 = 0.15) (AltiTrainer; SMTEC, Nyon, Switzerland). Recovery between the two tests (incremental exercise then time trial) was performed in normoxia.
Hypoxic Training
Subjects were paired according to their baseline V̇O2peak and randomly divided, in a double-blind, placebo-controlled manner, into two groups: eight subjects training in normoxia and the other nine in normobaric hypoxia (FIO2 = 0.15, corresponding to an altitude of approximately 2500 m). Subjects performed a total of 20 training sessions over 6 wk (3–4 sessions per week), each session lasting 60 min. Four different intensity profiles were alternately given to the subjects to keep motivation high (Table 1). In both groups (i.e., training in normoxia and training in hypoxia), subjects trained at the same relative workload, calculated from their individual Wmax previously determined in normoxia or hypoxia. To reach similar amounts of total work during training in both groups (because absolute workloads were lower during training in hypoxia), the subjects training in normoxia were furtively unloaded at the start and end of each session. During all training sessions, all subjects wore a face mask and inhaled into the air mixing system (AltiTrainer) connected to compressed air (delivering normoxia) or nitrogen (delivering hypoxia). By doing so, neither investigators nor subjects could identify the treatment. Subjects filled out a questionnaire after the training intervention, indicating that 47% guessed the right training group they were in.
Skeletal Muscle Sampling, Preparation, and High-resolution Spirometry
Skeletal muscle biopsies were obtained from the vastus lateralis muscle at baseline and after the 6 wk of endurance training. Samples were collected under local anesthesia using the Bergström technique with a needle modified for suction. The biopsy was immediately dissected free of fat and connective tissue and divided into sections for measurements of mitochondrial respiration, as reported elsewhere. All biopsies were taken approximately 48 h after the last bout of exercise. Some of the samples were collected in the morning, and others were collected in the afternoon, but all biopsies were collected under the same standardized conditions. The procedures are also described in the present article (see Text, Supplemental Digital Content 1, http://links.lww.com/MSS/A440, which describes the procedures for mitochondrial respiration measurements).
Total Hemoglobin Mass
Total hemoglobin mass (Hbmass) was measured with a carbon monoxide (CO) rebreathing technique, as previously described. Each time, the subject would come to the laboratory and first rest for 20 min in a semirecumbent position. Thereafter, 2 mL of blood was sampled from an antecubital vein via a 20-gauge catheter and analyzed immediately in quadruplicate for i) percent carboxyhemoglobin and Hb concentration ([Hb]) using a hemoximeter (ABL800; Radiometer, Copenhagen, Denmark); and ii) hematocrit (Hct) with the micromethod (4 min at 13,500 rpm). After baseline collection and control, the subject breathed 100% O2 for 4 min to flush the nitrogen from the airways. The breathing circuit (previously flushed with O2) was then closed, and a bolus (1.5 mL·kg) of 99.997% chemically pure CO (CO N47; Air Liquide, Paris, France) was administrated into the now closed rebreathing apparatus. The subject rebreathed this gas mixture for 10 min. At the end of the rebreathing period, an additional 2-mL blood sample was obtained and analyzed as before. The change in percent carboxyhemoglobin between the first and second measurement was used to calculate Hbmass, taking into account the amount of CO that remained in the rebreathing circuit at the end of the procedure (2.2%). Total RCV, blood volume, and plasma volume were derived from measures of Hbmass, [Hb], and Hct. All CO rebreathing tests were performed by the same researcher. Baseline and postendurance training values reported here correspond to the average of duplicate measurements conducted on separate days within these testing sessions. The coefficient of variation for Hbmass, assessed from duplicate measures and expressed as the percent typical error (i.e., SD of difference scores/√2), was 3.3% at baseline and 2.4% after endurance training. Of note, during baseline measurements, duplicate Hbmass recordings differed by 122 g in one subject. Without including this result, the typical error of Hbmass would have been 2.5% at baseline.
Statistics
The Kolmogorov–Smirnov test was applied to examine the normality in the distribution of data. The Bartlett test was used to evaluate the uniformity of variance between conditions. Resting, maximal exercise, and endurance performance data were analyzed using a two-way ANOVA with repeated measures, which included one between-subject (group, i.e., normoxic training and hypoxic training) and one within-subject (timing, i.e., pretraining and posttraining) factor. When necessary, differences between values obtained pre- and posttraining for a particular group were analyzed using Student's paired t-test. The effect of acute hypoxia on exercise parameters for a particular group was analyzed using Student's paired t-test. Statistics were done with the StatView software, version 5.0. (SAS Institute, Cary, NC). The values are reported as arithmetic means ± SD. A P value of <0.05 was considered significant.