Single Leg Cycling Offsets Reduced Muscle Oxygenation in Hypoxic Environments

intensity of large muscle mass exercise declines at altitude due to reduced oxygen delivery to active muscles. The purpose of this investigation was to determine if the greater limb blood ow during single-leg cycling prevents the reduction in tissue oxygenation observed during traditional double-leg cycling in hypoxic conditions. Ten healthy individuals performed bouts of double and single-leg cycling (4, four-minute stages at 50–80% of their peak oxygen consumption) in hypoxic (15% inspired O 2 ) and normoxic conditions. Heart rate, mean arterial pressure, femoral blood ow, lactate, oxygenated hemoglobin, total hemoglobin, and tissue saturation index in the vastus lateralis were recorded during cycling tests. Femoral blood ow (2846 912 mL/min) and oxygenated hemoglobin ( 2.98 3.56 au) during single-leg cycling in hypoxia were greater than double-leg cycling in hypoxia (2429 835 mL/min and 6.78 3.22 au respectively,p

conditions. Heart rate, mean arterial pressure, femoral blood ow, lactate, oxygenated hemoglobin, total hemoglobin, and tissue saturation index in the vastus lateralis were recorded during cycling tests. Femoral blood ow (2846 912 mL/min) and oxygenated hemoglobin ( 2.98 3.56 au) during single-leg cycling in hypoxia were greater than double-leg cycling in hypoxia (2429 835 mL/min and 6.78 3.22 au respectively,p 0.01). In addition, tissue saturation index was also reduced in the double-leg hypoxic condition (60.2 3.1% ) compared to double-leg normoxic (66.0 2.4%,p= 0.008) and single-leg hypoxic (63.3 3.2,p< 0.001) conditions. These data indicate that while at altitude, use of reduced muscle mass exercise can help offset the reduction in tissue oxygenation observed during larger muscle mass activities allowing athletes to exercise at greater limb/muscle speci c intensities. Keywords: blood ow; hypoxia; small muscle mass exercise; tissue oxygen saturation; muscle oxygenation; cycling; tissue perfusion; cardiovascular 1. Introduction Historically, endurance athletes have attempted to improve sea level performance through a variety of different means. The idea that training at altitude can augment sea level endurance performance gained widespread acceptance among athletes [1–8]. However, there has been debate over whether there are additional bene ts to altitude training [9–13] due to exercise intensity limitations imposed by reduced blood oxygen saturation at altitude as well as debate on the mechanisms responsible for such potential enhanced endurance performance [14]. As a result, the live high–train low paradigm [15–17] is a more optimal method to induce the favorable cardiovascular, metabolic, and respiratory adaptations of altitude exposure while circumventing decreased training intensity associated with exercise at altitude. However, for many athletes who live at altitude, the live high–train low paradigm is not feasible due to time constraints, nancial resources, and easily accessible training sites at lower altitudes. Thus, to optimize performance for these individuals it is imperative to nd an alternative way to train at sea level exercise intensities while living at altitude. Int. J. Environ. Res. Public Health2022,19, 9139.

altitudes. Thus, to optimize performance for these individuals it is imperative to nd an alternative way to train at sea level exercise intensities while living at altitude. Int. J. Environ. Res. Public Health2022,19, 9139.

Int. J. Environ. Res. Public Health2022,19, 9139 2 of 15 Single-leg cycling is a reduced muscle mass exercise that has been used as both a rehabilitative as well as training modality. Single-leg cycling has been reported to generate greater leg-speci c work rates [18–23] due to greater blood ow and oxygen delivery to active muscles [24]. This increased capacity to perform limb-speci c work or power during single-leg cycling and other reduced muscle mass activities (i.e., knee extension) leads to greater muscle speci c adaptations in highly trained and diseased populations [19,25–27] compared to double-leg cycling and other larger muscle mass training modalities. Thus, elevated limb speci c blood ow associated with single-leg cycling may also bene t exercise training at altitude. It has previously been reported that blood saturation, VO2peak, and peak power are reduced in single-leg cycling in hypoxia [28], however, it is still unknown if single-leg cycling can be used to at least partially offset the likely greater reduction in blood saturation and subsequent performance normally seen with traditional double-leg cycling. If so, this will allow athletes to exercise at the greater muscle-speci c work rates typically associated with sea level training. Therefore, the purpose of this study was twofold. In the rst study, we wanted to compare the physiological responses to double-leg cycling in normoxic and hypoxic conditions. We then wanted to assess whether transitioning to single-leg cycling in the hypoxic condition would minimize any observed reductions in tissue oxygenation. We hypothesized that tissue oxygen saturation would be reduced during submaximal double- leg cycling in hypoxia compared to normoxia. We also hypothesized that single-leg cycling will result in a greater hyperemic response in the active limb, thereby minimizing the reduction in tissue oxygenation in hypoxia. In the second study, we hypothesized that similar to submaximal exercise, a short bout of high-intensity exercise in hypoxia would compromise tissue oxygenation and reducing the muscle mass via single-leg cycling would restore tissue oxygenation similar to that observed during normoxia. 2. Materials and Methods 2.1. Participants A statistical power analysis was performed for sample size estimation using G*Power statistical power

the second study, we hypothesized that similar to submaximal exercise, a short bout of high-intensity exercise in hypoxia would compromise tissue oxygenation and reducing the muscle mass via single-leg cycling would restore tissue oxygenation similar to that observed during normoxia. 2. Materials and Methods 2.1. Participants A statistical power analysis was performed for sample size estimation using G*Power statistical power software (G*Power, Heinrich-Heine-University, Dusseldorf, Germany). The effect size for blood ow and hemoglobin was considered to be large (0.40) based on Cohen's f criteria with an alpha of (0.05) and a power of (0.80) the projected sample size needed with this effect size was 10. Ten healthy and recreationally active individuals were recruited to participate in phase 1 (5 males and 5 females; height:177.5 8.0 cm ; weight: 74.2 13.2 kg; VO2peak: 43.0 8.3 mL min kg; 25 3 years) as well as phase two (6 males and 4 females; height: 175.7 10 cm; weight: 74.9 15.4 kg; VO2peak: 44.4 7.5 mL min kg , age: 25 3 years) of this investigation. Participants selected for either phase were not obese, currently taking any medications, nicotine users, diagnosed with cardiovascular, respiratory, or metabolic diseases and were not exposed to an altitude above 2500 m within two months prior to participation in the study. Participants self- reported to be moderately active based on the American College of Sports Medicine (ACSM) exercise participation health screening guidelines [29]. For both phases of this study, participants were required to visit the laboratory on three separate occasions. This study was approved by the Kent State University Institutional Review Board (IRB log number 17–289). 2.2. Study Design Subjects were recruited to participate in either or both phases of this investigation. Each phase consisted of a preliminary testing session followed by two data collection sessions that were separated by 2–7 days. Phase one (Figure) consisted of three submaximal cycling conditions (double-leg normoxia, double-leg hypoxia and single-leg hypoxia) each with 4 levels of cycling intensity (3 4 repeated measures). Phase two (Figure) consisted of 3 maximal cycling conditions (double-leg normoxia, double-leg hypoxia and single-leg

session followed by two data collection sessions that were separated by 2–7 days. Phase one (Figure) consisted of three submaximal cycling conditions (double-leg normoxia, double-leg hypoxia and single-leg hypoxia) each with 4 levels of cycling intensity (3 4 repeated measures). Phase two (Figure) consisted of 3 maximal cycling conditions (double-leg normoxia, double-leg hypoxia and single-leg

Int. J. Environ. Res. Public Health2022,19, 9139 3 of 15 hypoxia). For both phases of this investigation the normoxic and hypoxic conditions were assigned in a counterbalanced order to visits 2 and 3.Int. J. Environ. Res. Public Health 2022, 19, x FOR PEER REVIEW 3 of 16 consisted of 3 maximal cycling conditions (double-leg normoxia, double-leg hypoxia and single-leg hypoxia). For both phases of this investigation the normoxic and hypoxic con- ditions were assigned in a counterbalanced order to visits 2 and 3. Figure 1. Timeline for phase 1 and 2. 2.3. Visit 1: Prescreening and Baseline Testing During the first session participants completed an informed consent and a health his- tory questionnaire. Subjects then performed a submaximal and maximal cycling protocol on a Velotron cycle ergometer (Racer Mate, Seattle, Washington, USA). The submaximal protocol consisted of 4 four-minute stages at 40, 80, 120, 160 watts (W) [24,30,31]. Follow- ing a 10-min recovery, subjects then performed a maximal cycle ergometer test that began at a work rate of 60 W for two minutes and increased 25 W every minute until the partic- ipant reached volitional fatigue. Within this study, these submaximal and maximal tests were used to quantify subject fitness (VO 2 peak) and prescribe the cycling workloads for the phase one protocol [30,32,33]. Oxygen consumption was measured during both cy- cling tests via Parvo-Medics metabolic cart (Parvo-Medics, Sandy, Utah, USA). These two tests were used to quantify subject fitness (VO 2 peak) and prescribe the cycling workloads for the phase one protocol. 2.4. Phase 1: Visits Two & Three Visits two and three were identical with the exception that one occurred in normoxic conditions and the other occurred with 15% inspired O 2 via hypoxia chamber (Altitude Control Technologies, Lafayette, CO, USA) simulating an altitude of 2740 m. Upon arrival into the laboratory, participants were seated in the hypoxia chamber, which was either turned on or off depending on the condition, and rested quietly for 30 min. During this time near infrared spectroscopy (NIRS) electrodes were secured to the skin over the vastus lateralis (approximately 15 cm above the

Lafayette, CO, USA) simulating an altitude of 2740 m. Upon arrival into the laboratory, participants were seated in the hypoxia chamber, which was either turned on or off depending on the condition, and rested quietly for 30 min. During this time near infrared spectroscopy (NIRS) electrodes were secured to the skin over the vastus lateralis (approximately 15 cm above the proximal border of the patella and five centime- ters lateral to the midline of the thigh) (Artinis Medical Systems, Oxymon MkIII, The Netherlands). Following the 30-min acclimation period heart rate, blood pressure, SaO2 and lactate were measured. Additionally, NIRS was used to obtain a measurement of muscle oxygenated hemoglobin, total hemoglobin as well as tissue saturation index (TSI) which is the ratio of oxygenated hemoglobin to total hemoglobin. The experimental protocol consisted of two 16-min bouts of either single or double- leg cycling presented in random order. The 16-min bouts were composed of four 4-min stages. The workloads for the double-leg cycling were set to achieve 50%, 60%, 70% and 80% of the subject’s VO 2 peak [24,30,31]. To maintain limb specific work rates, the work- loads during single leg cycling were half that of double-leg. Subjects were instructed to maintain a pedaling rate of 80 rpm based on visual feedback from the Velotron software. There was a 30-s break between each stage to allow for femoral blood flow recording and a 15-min break between the single and double-leg protocols. During each stage, changes Figure 1.Timeline for phase 1 and 2. 2.3. Visit 1: Prescreening and Baseline Testing During the rst session participants completed an informed consent and a health history questionnaire. Subjects then performed a submaximal and maximal cycling protocol on a Velotron cycle ergometer (Racer Mate, Seattle, Washington, USA). The submaximal protocol consisted of 4 four-minute stages at 40, 80, 120, 160 watts (W) [24,30,31]. Following a 10-min recovery, subjects then performed a maximal cycle ergometer test that began at a work rate of 60 W for two minutes and increased 25 W every minute until the participant reached volitional fatigue. Within this study, these submaximal and maximal

USA). The submaximal protocol consisted of 4 four-minute stages at 40, 80, 120, 160 watts (W) [24,30,31]. Following a 10-min recovery, subjects then performed a maximal cycle ergometer test that began at a work rate of 60 W for two minutes and increased 25 W every minute until the participant reached volitional fatigue. Within this study, these submaximal and maximal tests were used to quantify subject tness (VO2peak) and prescribe the cycling workloads for the phase one protocol [30,32,33]. Oxygen consumption was measured during both cycling tests via Parvo-Medics metabolic cart (Parvo-Medics, Sandy, Utah, USA). These two tests were used to quantify subject tness (VO2peak) and prescribe the cycling workloads for the phase one protocol. 2.4. Phase 1: Visits Two & Three Visits two and three were identical with the exception that one occurred in normoxic conditions and the other occurred with 15% inspired O 2 via hypoxia chamber (Altitude Control Technologies, Lafayette, CO, USA) simulating an altitude of 2740 m. Upon arrival into the laboratory, participants were seated in the hypoxia chamber, which was either turned on or off depending on the condition, and rested quietly for 30 min. During this time near infrared spectroscopy (NIRS) electrodes were secured to the skin over the vastus lateralis (approximately 15 cm above the proximal border of the patella and ve centimeters lateral to the midline of the thigh) (Artinis Medical Systems, Oxymon MkIII, The Netherlands). Following the 30-min acclimation period heart rate, blood pressure, SaO2 and lactate were measured. Additionally, NIRS was used to obtain a measurement of muscle oxygenated hemoglobin, total hemoglobin as well as tissue saturation index (TSI) which is the ratio of oxygenated hemoglobin to total hemoglobin. The experimental protocol consisted of two 16-min bouts of either single or double-leg cycling presented in random order. The 16-min bouts were composed of four 4-min stages. The workloads for the double-leg cycling were set to achieve 50%, 60%, 70% and 80% of the subject's VO2peak [24,30,31]. To maintain limb speci c work rates, the workloads during single leg cycling were half that of double-leg. Subjects were instructed to maintain a

or double-leg cycling presented in random order. The 16-min bouts were composed of four 4-min stages. The workloads for the double-leg cycling were set to achieve 50%, 60%, 70% and 80% of the subject's VO2peak [24,30,31]. To maintain limb speci c work rates, the workloads during single leg cycling were half that of double-leg. Subjects were instructed to maintain a pedaling rate of 80 rpm based on visual feedback from the Velotron software. There was a 30-s break between each stage to allow for femoral blood ow recording and a 15-min break between the single and double-leg protocols. During each stage, changes in oxyhemoglobin, total hemoglobin and TSI were recorded continuously. Heart rate, blood pressure, SaO2 and lactate were recorded during the last minute of each stage. Finally, femoral artery blood velocity and vessel diameter were measured at the end of each stage

Int. J. Environ. Res. Public Health2022,19, 9139 4 of 15 using a Logiq 7 Doppler/ultrasound machine with an M12 linear transducer (General Electric Medical Sytems, Milwaukee, WI, USA). Speci cally, following the completion of each four-minute stage the participants were instructed to immediately extend their active leg and rest it on a box next to the ergometer. While participants remained seated, the ultrasound image of the femoral artery was obtained and blood ow was measured within 4–5 s of pedaling cessation. Marking the location for probe placement on the skin following baseline measurements aided in the quick transition from exercise cessation to probe placement. Once the vessel image was obtained, blood ow was measured for 10 s and then the participant began the next stage of cycling. Femoral blood ow was calculated in milliliters per minute based on blood velocity and arterial diameter utilizing the following equation: Blood ow = Vmean (vessel diameter/2) 2 60. During the single-leg cycling a modi ed wooden box was placed directly next to the unoccupied crank arm in order to allow the participant to rest their inactive leg. A 10-kg counterweight was attached to a spindle on the crank arm opposite the active cycling leg which minimized the biomechanical differences between double-leg and single-leg cycling. Speci cally, the counterweight assisted the active limb on the pedal upstroke, negating the need to recruit hip exor muscles, while maintaining a smooth cycling motion similar to double-leg cycling [24,34]. Finally, only the right leg was used for single-leg cycling primarily due to the small space inside our hypoxia chamber and placement of ultrasound machine in relation to cycle ergometer which allowed easy access to the right femoral artery for blood ow measurements. For control purposes, the same researcher performed all of the femoral artery blood ow measurements. 2.5. Phase 2: Visits 2 and 3 Phase 2 focused on shorter bouts of maximal cycling exercise. These two visits were identical to each other with the exception that one was performed in normoxic conditions while the other was performed in hypoxic conditions. Upon arrival into the laboratory, participants were

researcher performed all of the femoral artery blood ow measurements. 2.5. Phase 2: Visits 2 and 3 Phase 2 focused on shorter bouts of maximal cycling exercise. These two visits were identical to each other with the exception that one was performed in normoxic conditions while the other was performed in hypoxic conditions. Upon arrival into the laboratory, participants were instructed to remain seated in the hypoxia chamber (which was turned on or off depending on condition) and rested quietly for 30 min to allow for acclimation. During this time, near infrared spectroscopy (NIRS) electrodes were secured to the skin over the vastus lateralis. Following the initial warm-up, they performed either a double-leg or single-leg 30 s maximal Wingate test which were assigned to the subjects in a counter balanced order. A 15-min recovery period separated the double-leg and single-leg Wingate tests. The resistance on the ywheel was set at 9% body weight for the double-leg trial and 5.4% body weight for the single-leg trial for both sexes (note: as single-leg Wingate has never been reported in adults, the 5.4% resistance was based on pilot studies within our lab in which 60% of the double-leg resistance [0.090 0.60 = 0.054] produced max power) [2,35–40]. The counterweight was utilized for the single-leg trial as described above. Throughout each 30 s Wingate protocol, tissue oxygenation, oxygenated hemoglobin, total hemoglobin, and TSI were recorded. Full recovery of the participant was determined by feedback from subjects and ensuring lactate, heart rate, and tissue oxygenation were back to baseline levels. 2.6. Data Analysis The main goal of this investigation was to determine if the increased blood ow during single-leg cycling in hypoxia (SLH) can improve tissue oxygenation compared to double-leg cycling in hypoxia (DLH) such that it would be similar what is observed during double-leg cycling in normoxic conditions (DLN). Thus, the single-leg trial in normoxia, although performed to maintain consistency between the hypoxia and normoxia trials, was deemed not relevant and to maintain focus of the paper those data are not reported. The dependent variables that were assessed during the submaximal cycling protocols were

This study investigates the effects of single-leg cycling on muscle oxygenation in hypoxic conditions.