To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.
Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.
Physical activity is defined as any bodily movement produced by skeletal muscles that require energy expenditure1. Exercise is a physical activity that involves repetitive bodily movement done to improve or maintain one or more components of physical health2. At one time, physical activity was not recommended for those who were seriously ill. For individuals with cancer, heart failure, or even for those who were pregnant, bed rest was preferred over physical activity. Clinical practice has since drastically changed, as the benefits of exercise on overall health are becoming undeniable3. Regular exercise has been shown to help reduce cardiovascular disease risk, all-cause mortality, cancer risk and hypertension, improve blood sugar control, facilitate weight loss or maintenance, and prevent bone and muscle loss4,5,6,7,8.
The extensive benefits of exercise have now led many to utilize exercise as a type of "medicine" and an alternative or adjunct treatment option for a variety of conditions3. Shulman et al. demonstrated that a combination of treadmill and resistance exercise could result in improvements in gait speed, aerobic capacity and muscular strength which could improve motor control and overall quality of life in patients with Parkinson's disease9. In heart failure patients, exercise intolerance and inadequate pharmaceutical interventions contribute to a poor quality of life10. Initial results from heart failure patients undergoing exercise training in the HF-ACTION trial demonstrated improvement in quality of life and reductions in hospitalizations and mortality11. Additionally, the application of exercise to alter the cardiotoxic effects of anthracycline-containing chemotherapy (e.g., doxorubicin) has demonstrated that regardless of when it is initiated with respect to the patients chemotherapy administration (before, during or after), exercise can provide beneficial effects such as reducing the decline in aerobic capacity, attenuating the left ventricular dysfunction and reducing oxidative damage12.
The benefits of exercise in health and wellness are not just in its application as a medicine/treatment, but also as a diagnostic tool. Exercise testing is, for example, used to diagnose exercise intolerance, ischemia in the heart, or to understand the cause of shortness of breath13. Perhaps more importantly, exercise testing may be utilized to identify subclinical dysfunction. The human body is in most situations "overbuilt," such that dysfunction or pathophysiology can often remain hidden and unapparent to an individual for months or years. This observation may explain why conditions such as pulmonary arterial hypertension or pancreatic cancer can silently increase in severity such that by the time symptoms are noticed, these conditions tend to be very advanced and extremely difficult to treat2. In some of these situations, exercise testing can provide a stress stimulus to the body which increases demand above that of daily living and at times can identify dysfunction (cardiac, respiratory, metabolic) that was not seen at rest, helping to diagnose a disease and begin treatment earlier.
In order to fully maximize the therapeutic and diagnostic potential of exercise, standardized methods to quantify the responses to physical activity are needed to accurately assess the contributions of exercise to overall immune health. Variations in workload, inclination, duration, type of exercise, and the timing of sample collection can all influence measurements of physiological responses. Here, we outline methods for maximal and submaximal endurance exercises to gather physiological data while collecting samples for biological responses. This methodology was used to understand how acute exercise affected the distribution and frequency of leukocyte populations in peripheral blood14 by measuring immune cell populations at various time points before and after exercise by flow cytometry with 10-color flow protocols that permit the quantification of all major leukocyte subsets simultaneously15. The following protocol can be used as a standardized method for two distinct exercise regimens for measuring physiological and biological responses to exercise.
The protocol was approved by the Mayo Institutional Review Board and conformed to the Declaration of Helsinki. All participants provided written informed consent before participating in the testing described.
1. Calibration and Setup of Metabolic Cart
2. Pulmonary Function Test (PFT)
NOTE: The pulmonary function test methods described are a brief summary of those published by the American Thoracic Society and European Respiratory Society, for additional details please refer to their publications16,17.
3. Exercise Tests
4. Blood Analysis
The application of maximal or submaximal endurance exercise testing provides a stimulus or stressor in which the body responds to meet the increased physiological demands. Various modes of exercise can be used to compare the physiological and biological responses to a particular exercise by itself or when a drug/intervention is used, or to evaluate the differences in responses between different exercise loads. Maximal and endurance exercise loads differ in the duration (short/long respectively) and intensity (high/low respectively), while the mode, (i.e., cycling), is held constant. When designing a study with exercise testing, it is important to establish what the goals of the use of exercise are and what type of response is desired. Table 1 highlights the differences and similarities between submaximal endurance and maximal exercise testing, but researchers also need to be cognizant of the effects different modalities of exercise will have on the parameters being evaluated. In a maximal exercise test, where demand or exercise intensity is continually increasing with increases in workload (resistance/wattage on a bike or speed and/or grade on a treadmill) the cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production) also continuously increase (Figure 1A). In contrast, during a submaximal endurance exercise test the demand is increased from that at rest, but is raised to fixed exercise intensity. As such, the cardiopulmonary response has an initial increase, but then plateaus as the body adapts to meet the consistent demand (Figure 1B). The difference in intensity and demand between maximal and submaximal endurance exercise testing is also apparent when reviewing the change in rating of perceived exertion (RPE) and the respiratory exchange ratio (RER) over the respective exercise bout which estimates the fuel being used to supply the body with energy. In a maximal exercise test RPE and RER will steadily increase until the end of the test (Figure 2A), where as in a submaximal endurance exercise test these parameters will plateau (Figure 2B).
Although not required, it can be beneficial to perform a pulmonary function test before performing an exercise test. Exercise elicits a cardiac and pulmonary response and the performance during the exercise test can be limited by metabolic function and the ability of the heart, lungs or both to respond. When evaluating if there is a pulmonary limitation, it is helpful to know resting pulmonary function which can identify obstructive or restrictive limitations through the slow vital capacity (SVC) and forced vital capacity (FVC) maneuvers. Performing the maximal voluntary ventilation (MVV) maneuver to determine ventilatory capacity is useful as this can then be utilized to determine how much ventilatory reserve is present or if the individual is encroaching upon their ventilatory limits. However, this value can also be estimated from the FEV1. Before performing pulmonary function testing, one should review the standardized methods for spirometry provided by the American Thoracic Society and European Respiratory Society16,17.
Figure 1: Gas Exchange and Heart Rate Data for Maximal and Submaximal Endurance Exercise Tests. Physiological changes in response to increasing workload (resistance in watts) in maximal test (A) and changes observed in an endurance test (over time) (B). Panels show the change in oxygen consumption (VO2, open downward triangles), carbon dioxide production (VCO2, black triangles) on the left y-axis and ventilation (VE, black circles) and heart rate (HR, grey circles) on the right y-axis. Peak oxygen consumption (VO2 Peak) and test duration or endurance submaximal workload are listed on each panel figure. Please click here to view a larger version of this figure.
Figure 2: Parameters of Exercise Intensity for Maximal and Endurance Exercise Tests
Two panels show the change in the rating of perceived exertion (RPE, asterisk) on the left y-axis and respiratory exchange ratio (RER, black downward triangle) on the right y-axis in response to increasing work (Watts) for maximal exercise test (A) and time (min) for the submaximal endurance test (B) on the x-axis. Please click here to view a larger version of this figure.
Maximal Exercise Test | Similarities | Endurance Submaximal Exercise Test |
– Goal duration 10–20 min | – HR continuously monitored | – Duration 30+ min |
– Increasing intensity: ramping or stages | – BP measured at regular intervals | – Steady state, desired intensity chosen and held |
– Gas exchange continuously monitored | – RPE measured | – Intermittent gas exchange monitoring |
– HR, VO2, VCO2 and VE steadily increasing with increasing workload | – Pulse oximetry continuously monitored | – HR, VO2, VCO2 and VE plateau and workload is decreased if start to rise as goal is to keep these steady |
– RER is ≥ 1.1 | – RER stays below < 1.0 | |
– At end measurements will be the maximum that the individual’s body can produce (HR, VO2, VCO2, VE, workload, etc.) | – At the end measurements will be the percentage of the maximum that the individual’s body can produce (HR, VO2, VCO2, VE, workload, etc.). Percentage is dictated by the intensity of submaximal exercise and/or the duration | |
– To know what percentage of maximum it is a maximal exercise test would be needed to be performed usually at an earlier visit | ||
VO2: oxygen consumption; VCO2: carbon dioxide production; VE: ventilation; HR: heart rate; RER: respiratory exchange ratio |
Table 1: Comparison of Maximal and Submaximal Endurance Exercise Tests. The table summarizes the differences and similarities between the two exercise tests described.
There is great potential for exercise to be incorporated as an adjunct/alternative therapeutic tool. Indeed, an emerging body of evidence strongly suggests that physical activity promotes good health. The use of exercise as a medicine or diagnostic tool would require an understanding of the right amount or "dose" of exercise to achieve the desired effect. The optimal dose of exercise should be estimated, as too much exercise may be detrimental to improving health. As such, an exercise regimen may need to be tailored to each individual to achieve the optimal benefit from exercise. To that end, the variables that contribute to the nature of the diverse responses to exercise need to be understood and controlled. Therefore, standardized methodologies to exercise testing will be critical in moving the field forward.
The best method for normalizing exercise intensity for submaximal exercise testing continues to be the subject of debate. We chose to use 60% of the maximal workload achieved, but percentage of VO2max/peak, HRmax or HRRmax are commonly used for prescribing exercise training intensity zones19. More recently, other methods have been suggested as being more effective at normalizing exercise intensity for research. One being the percentage delta concept, where the intensity is set to a specified percentage of the difference between the gas exchange threshold and VO2max and has been shown to provide more consistent between-subject responses to endurance submaximal exercise testing than using a percentage of VO2max20. A second method for cycling excise testing is critical power (CP) which describes power output that corresponds to the fatigue threshold. At this point cardiopulmonary and metabolic responses are most synchronized or unified. When exercise is performed below this threshold, peripheral fatigue does not limit the duration the exercise can be performed for, and exercise intensity can be stabilized. On the other hand, above CP, the amount of work that can be done or W' can be identified and the duration until W' is exhausted can be predicted21. The best choice for how to determine the submaximal exercise intensity remains yet to be determined, but many in the exercise physiology field are moving away from the older methods and moving towards one of the newer methods described. The protocol chosen depends upon the study and the primary outcomes being evaluated. Additionally, in this study intermittent monitoring of gas exchange was chosen to make the test more comfortable for the participants as breathing on a mouthpiece for long periods is uncomfortable due to mouth dryness. Saliva can accumulate and holding the mouthpiece in the mouth can be tiring for the jaws. Since the primary outcome was a change in peripheral blood leukocytes and not a change in cardiopulmonary response to submaximal endurance exercise, intermittent monitoring of gas exchange to ensure that the exercise test remained at steady-state was sufficient.
We have outlined standardized exercise protocols, but additional steps can be taken preceding the exercise testing to further improve the consistency and reproducibility of exercise testing results. For example, have the same technician perform all of the blood pressure measurements for a particular study, or at minimum, have the same technician measure a subject over multiple repeat tests. Second, a proper calibration of all test equipment, especially the metabolic analyzer, should be performed prior to each experiment. Finally, the variability of the subject population and how this will alter the individual response and comparisons between individuals should be considered and minimized. This can be mitigated in several ways through restricting the use of stimulants (i.e., caffeine), and controlling food intake and exercise prior to testing and also ensuring that the subjects is well rested. The testing conditions (equipment, room temperature, time of day, etc.) should be kept consistent if tests are going to be repeated. In some scenarios, having female participants complete testing on a particular phase of their menstrual cycle (e.g., early follicular phase) is also an important control. Additionally, the researcher will need to decide if they will allow supplements and medications to be taken, as these can alter the response to exercise. While there may be additional variables to control for in a particular study, we strongly recommend that these steps be incorporated into any study design involving exercise testing.
The exercise regimens described here can be utilized to study physiological responses to acute exercise. We have previously used this methodology to understand immunological changes in healthy individuals in the two different exercise regimens14. We collected blood samples prior to exercise testing with three blood samples collected at different time points after exercise. While both maximal and endurance exercise regimens led to a rapid accumulation of several leukocyte populations, the maximal regimen lead to a greater increase of most leukocyte subpopulations immediately after the testing was performed. We also found that CD56+CD16+ natural killer cells increased the most immediately after exercise, but CD15+ granulocytes had a delayed response by peaking at three hours post exercise. As it is well known that peripheral blood leukocytes rapidly accumulate into circulation following exercise (reviewed by Freidenreich and Volek22), our study demonstrated that the kinetics of mobilization are quite different and cell type specific. Natural killer (NK) cells and CD8+ cytotoxic T cells appear to be the most influenced upon exercise23, but other populations including myeloid cells and B cells also increase to some degree. While many studies have focused on acute effects of single exercise events, longitudinal training-based exercise regimens are likely needed to provide additional insight into how exercise affects long-term immunological performance.
The protocols described here provide a standardized methodology to incorporate exercise regimens for biologic and physiologic responses. These protocols can be easily modified for both single exercise tests as well as long-term longitudinal multiple testing. Physiologic measurements may include, but are not limited to, heart rate, blood pressure, oxygen consumption, and body mass index (BMI). Biologic responses can be measured from a variety of specimens including peripheral blood, saliva, urine, and sweat. From these samples, multiple concurrent analyses can be performed via flow cytometry of cellular composition, proteomics analyses, gene expression arrays, or other types of biochemical and molecular approaches. In addition to understanding changes in the composition of peripheral blood leukocytes, others have looked at plasma markers of inflammation24, cytokines25, and how training regimens may be used to alter exercised-induced changes26. Taken together, standardized protocols allow the measurement of physical activity of different durations and intensities with associated physiological parameters in a defined manner.
The authors have nothing to disclose.
This study was funded by the Mayo Clinic Department of Laboratory Medicine and Pathology and other various internal sources.
Metabolic cart/portable system | MCG Diagnostics | Mobile Ultima CPX System | The flow calibration syringe, and calibration gases should come with system. There are numerous possible options/alternatives. |
Pulmonary function software (Breeze Suite) | MCG Diagnostics | Software used will depend on the metabolic system | |
Upright cycle ergometer | Lode ergoline | 960900 | Numerous possible options/alternatives |
12-Lead ECG | GE Healthcare | CASE Exercise Testing System | Used for 12 lead ECG capture, control bike. Having a full 12-lead is ideal for maximal exercise test so can monitor for arhythmias, but alternative for just HR would be a wireless chest strap heart rate monitor |
Pulse oximeter | Masimo | MAS-9500 | Usually multiple probe options: finger, forehead, ear lobe. Usually avoid finger as tight handlebar grip can cause measurement inaccuracies |
Pneumotach (preVent Flow Sensor) | MCG Diagnostics | 758100-003 | Alternative systems can use a turbine |
Nose piece (disposable) | MCG Diagnostics | 536007-001 | Numerous possible options/alternatives |
Mouthpeice with saliva trap | MCG Diagnostics | 758301-001 | Suggest filling the saliva trap with paper towel/gauze and tape cap to limit dripping |
Headband | Cardinal Health | 292866 | Used to secure the forehead pulse oximeter and the lines for the cart |
Stethescope | 3M Littman | 3157SM | Numerous possible options/alternatives |
Blood pressure cuff | HCS | HCS9005-7 | Cuff size will depend on the population planning to test |
ECG Electrodes | Cardinal Health | M2570 | only needed with lead based ECG/HR monitoring |
K2EDTA tube 5mL | Becton Dickinson | 368661 | |
*The table provides a list of the supplies and equipment utilized in this protocol and comments related to the equipment. Brand name/company is provided, but the use of other brands will not affect the results, key is to keep it consistent throughout testing in a particular study. |