This study was funded by the Mayo Clinic Department of Laboratory Medicine and Pathology and other various internal sources.

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
<ol>
	<li>Flow and volume (pneumotach) calibration 
	NOTE: Specific materials and equipment are listed in the Materials Table.
	<ol>
		<li>Open the pulmonary function and gas exchange software to calibrate for the test.</li>
		<li>Click the &#8220;calibrate&#8221; button to open the calibration window. At the bottom of the calibration window, note the room temperature, barometric pressure, and the humidity &#8212; ensure that these values are accurate using a barometer.</li>
		<li>Insert the umbilical and sample line into the pneumotach and then insert the pneumotach into the 3 L syringe.</li>
		<li>Before starting calibration, click the &#8220;zero flow&#8221; button to ensure that there is no flow going through the syringe.</li>
		<li>Click &#8220;start&#8221; to begin the calibration. Withdraw first, then inject. Repeat 4 more times (5 total), except at different flow rates each time. Maintain constant flow rate for each respective withdrawal/injection, then vary flow rate for the next iteration.</li>
	</ol>
	</li>
	<li>Gas calibration
	<ol>
		<li>Attach sample line from respiratory mass spectrometer/gas analyzer/metabolic cart to the calibration/home port on the system.</li>
		<li>In the pulmonary function software, select the O2/CO2 analyzers tab in the calibrate window. Open the reference (room air 21% O2, 0.04% CO2) and calibration (12% O2 and 5% CO2) gas tanks.</li>
		<li>Select the calibrate button. 
		NOTE: The software will operate the solenoid valves in the metabolic cart system to switch between the reference and calibration gases being sampled at the calibration port. From this, the software will evaluate for any offset, sampling the delay due to sample line length and the 2&#8211;90% response time. A green &#8220;Calibration Successful&#8221; message will appear when calibration is complete.</li>
	</ol>
	</li>
	<li>Final Setup
	<ol>
		<li>Remove the sample line from the gas calibration port and insert it back into the pneumotach. Then attach a mouthpiece onto the pneumotach.</li>
		<li>Wait for the subject to arrive for exercise test.</li>
		<li>Upon arrival of the subject, inform them about the study and what their involvement will be, review the consent form with them and have him/her sign it, and as the visit proceeds continually discuss with them what (s)he will be doing next and provide explanation of the procedures before doing them. 
		NOTE: Inclusion and exclusion criteria will vary based on the purpose of the exercise testing, for this study those recruited were healthy, non-smokers, with no known cardiopulmonary or immune disease and not taking any steroids or immune modulating drugs.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Slow vital capacity (SVC) maneuver
	<ol>
		<li>Instruct the subject to be seated with straight back and feet flat on the floor with legs uncrossed.</li>
		<li>Instruct the subject to put his/her mouth around the mouthpiece, and bite down; also fit the subject with a nose clip to seal off the nasal cavity.</li>
		<li>Start the maneuver on the software. When beginning the maneuver, instruct the subject to continue to breathe normally.</li>
		<li>Observe the subject&#8217;s tidal breathing and have him/her continue to breathe while waiting for him/her to reach a stable breathing pattern.</li>
		<li>Instruct the subject to inhale maximally and then exhale slowly. The subject will keep emptying his/her lungs until they can no longer exhale. This point will be apparent by a plateau in the flow tracing. At this point, instruct the subject to take a maximal breath in.</li>
		<li>Stop the measurement and instruct the subject to release the mouthpiece and remove the nose clip to take a break if needed. 
		NOTE: A minimum of three SVCs are performed. To meet American Thoracic Society standards, they must agree within 5% or 150 mL of each other for both the largest VC and inspiratory capacity (IC) values. Up to 4 maneuvers can be performed in order to obtain three that agree16,17.</li>
	</ol>
	</li>
	<li>Forced vital capacity (FVC) maneuver
	<ol>
		<li>Instruct the subject to grasp the mouthpiece and attach the nose clip while remaining in the same seated position.</li>
		<li>Start the maneuver on the software and instruct the subject to continue to breathe normally. Ensure the subject has established a stable breathing pattern with a minimum of four tidal breaths.</li>
		<li>Instruct the subject to inhale fully and rapidly, and then to immediately exhale (pause of &#60; 1 s) as quickly and forcefully as possible.</li>
		<li>Tell the subject/patient to continue to try and blast/push all the air out of his/her lungs reaching a full exhalation while remaining in an upright posture. This is seen as a plateau in the volume-time curve. Ask them to continue trying to exhale for as long as they can; ideally for 6 s.</li>
		<li>Once this is achieved, instruct the subject to take a maximal breath in and stop the maneuver on the software. 
		NOTE: The test should be repeated at least two more times &#8212; ensuring that they agree within 5% or 150 mL of each other for both the two largest FVC and FEV1 values. Up to 8 maneuvers can be performed in order to get two that agree.</li>
	</ol>
	</li>
	<li>Maximum voluntary ventilation (MVV) maneuver 
	NOTE: The goal of this maneuver is to have the subject move as much air as rapidly as they can. They will be coached to try to take as large breaths as possible while still breathing rapidly.
	<ol>
		<li>Instruct the subject to grasp the mouthpiece and attach the nose clip while maintaining the same seated position.</li>
		<li>Start the maneuver on the software. 
		NOTE: A countdown bar at the top will indicate the number of breaths required (usually three) before data collection/measurements begin.</li>
		<li>With one breath to go in the countdown, direct the patient to start breathing deeply and rapidly through the mouthpiece. They will continue this for 12 s.</li>
		<li>Encourage the patient throughout the procedure to breathe deeply and fast. If the subject is unable to continue, stop the test.</li>
		<li>At the end of 12 s, instruct the patient to resume normal breathing. They may feel light headed, so encourage them to sit back and take slow deep breaths. 
		NOTE: For repeatability the test should be performed a minimum of two times and the suggested variability should be less than 20%.</li>
	</ol>
	</li>
</ol>
3. Exercise Tests
<ol>
	<li>Electrode placement
	<ol>
		<li>Prepare the skin for the electrodes by shaving hair away from electrode placement site if present. Rub the site with an alcohol pad and then with an abrasive pad to remove any dead skin cells. 
		NOTE: Electrode can be placed once this is completed, but be sure that the electrode has adequate gel and is not dry.</li>
		<li>Equip the subject with electrodes for a 12-lead electrocardiogram using the following electrodes placement.
		<ol>
			<li>Position limb lead electrodes as follows: RA: right side subclavicular fossa; LA: left side subclavicular fossa; RL: right back just above posterior superior iliac spine; LL: left back just above posterior superior iliac spine</li>
			<li>Position precordial leads as follows: V1: right of sternum in the 4th intercostal space; V2: left of sternum in the 4th intercostal space (in line with V1); V3: left side directly between V2 and V4; V4: left side in the 5th intercostal space at the midclavicular line (typically under breast/nipple); V5: place horizontally with V4 on anterior axillary line (down from the edge of the armpit (anterior axillary fold of the upper arm); V6: place horizontally with V4 and V5 on the middle auxiliary line.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incremental maximal cycling test &#8212; Visit 1 
	NOTE: Performing a maximal exercise test&#160;comes with risks. The American College of Sports Medicine outlines how to identify individuals who are at higher risk for an adverse event during the test13. Those who would be considered to have significant risk have: known cardiovascular, pulmonary and/or metabolic disease; major symptoms: chest pain, shortness of breath (SOB) at rest or with mild exertion, dizziness or syncope, orthopnea, ankle edema, palpitations or tachycardia, intermittent claudication, known heart murmur, unusual fatigue or SOB with usual activities; or at least two of the cardiovascular disease risks factors: family history of myocardial infarction or sudden death, age (males &#8805; 45, women &#8805; 55), current smoker, sedentary lifestyle ( &#60; 30 min of moderate intensity physical activity 3 days a week for at least 3 months), obesity (BMI &#8805; 30 kg/m2), hypertension (Systolic blood pressure (SBP) &#8805; 140 mmHg or Diastolic blood pressure (DBP) &#8805; 90 mmHg), dyslipidemia (total cholesterol &#8805; 200 mg/dL; LDL &#8805; 130 mg/dL; HDL &#60; 40 mg/dL, or on lipid lowering medication), prediabetes (fasting blood glucose &#62; 100 mg/dL). All maximal exercise testing should be performed under the supervision of a health care professional trained in clinical exercise testing, with at least two people present, one to monitor the ECG and the other to be taking blood pressures and monitoring the patient. For those who are higher risk, a physician should also be present during the test, whereas in those that are at lower risk the test can be performed without a physician present; it is preferred to have the physician nearby and available immediately if needed. The personnel performing the test should have basic life support with an automated external defibrillator (AED) in the room and at least one or more performing personnel should have advance cardiac life support training. Those performing the test should know the plan for responding to a medical emergency and have the appropriate contact numbers.
	<ol>
		<li>Fit the subject to the bike making sure the seat and handlebars are positioned comfortably. 
		NOTE: The general suggestion for seat height is such that the leg has a slight bend at the bottom of the pedal stroke and the seat should be adjusted horizontally so that when the crank arm is parallel to the ground the knee should fall over the metatarsals which should be over the pedal spindle. The handlebars should be at the same height as the seat or slightly higher and close enough so that the subject has a slight bend in their elbows. The handlebar position will depend on the experience of the rider, more experienced cyclists will want to be more bent over, where are those who do not bike often will prefer a more upright position.</li>
		<li>Place a pulse oximeter on the subject&#8217;s forehead. Wipe the placement location with an alcohol wipe to remove any makeup or dirt, etc. and secure it to the forehead with a headband.</li>
		<li>Review the exercise test procedure with the subject. Inform the subject that they will need to stay breathing through the mouthpiece for the entire duration of the test, and breathe only through his/her mouth as the nose will be plugged with the nose clip. 
		NOTE: Using a mouthpiece and nose clip is not the only option; masks are available which cover the nose and mouth allowing the participant to breathe through either their mouth or nose18. Gas exchange metrics are continuously measured and recorded through the pulmonary function software. Heart rate (HR) and rhythm will be continuously monitored by the 12-lead electrocardiogram (ECG). Peripheral oxygen saturation (SpO2) will be continuously monitored with the pulse oximeter. These external signals (HR, SpO2) can be linked to the metabolic cart so that all measurements are documented together. If this isn&#8217;t possible, HR and SpO2 should be recorded on a worksheet every minute.</li>
		<li>Draw a 5 mL blood sample from the antecubital vein (baseline exercise blood draw).</li>
		<li>After 2 min of rest, begin data collection. Then, start the exercise protocol and instruct the participant to start&#160;pedaling. Ask them to reach a pedal rate between 60 and 80 rpms. 
		NOTE: For this study, the maximal exercise test protocol used was an initial workload of 50 W with 30 W increments every 2 min. The protocol used can vary depending upon the population and the goals of the test. For older individuals or patient populations, the first stage can be performed unloaded to allow the individuals to get their legs moving before resistance is added. In younger healthy individuals, this is usually not necessary as 50 W is a low enough workload to warm up. Pedaling at 0 W while maintaining the desired pedal rate is actually more challenging than starting with resistance from the beginning.</li>
		<li>Have an assisting technician measure blood pressure (BP) 1 min into each stage while a second technician assists with the test. Then ask the subject to rate his/her exertion level on the Borg Rating of Perceived Exertion (RPE) scale where 6 indicates that the exertion is perceived as easy (like (s)he is sitting/standing doing nothing) and 20 indicates that the perceived exertion is at the hardest work they can imagine doing18. Take a 12-lead ECG printout within the last 30 seconds of each stage.</li>
		<li>Continue the test until exhaustion of subject which is designated by having at least two of the following occur: when 60-80 rpm on the bike can no longer be maintained, the subject&#8217;s VO2 plateaus and does not increase with an increase in workload, his/her respiratory exchange ratio (RER) is equal to or greater than 1.1&#8211;1.2, and/or the subjects rating of perceived exertion (RPE) &#8805; 18.</li>
		<li>Stop the test if any of the following occur13: onset of angina or chest pain symptoms; drop in SBP of &#8805; 10 mmHg with an increase in work; excessive rise in BP: SBP &#62; 250 mmHg and/or DBP &#62; 115 mmHg; shortness of breath, wheezing, leg cramps, or claudication; signs of poor perfusion: light-headedness, confusion, nausea, cyanosis, cold or clammy skin; failure of HR to increase with increasing exercise intensity; change in heart rhythm with symptoms; subject requests to stop; subject vocalizes or severe fatigue is observed; testing equipment not functioning properly.</li>
		<li>Upon reaching exhaustion, proceed to the recovery phase: drop the resistance to the initial workload and instruct the subject to continue to cycle for another 2 min.</li>
		<li>Draw another 5 mL of blood from the patient (post exercise blood draw) via antecubital venipuncture.</li>
		<li>Direct the subject to return in 3 h, and 24 h following the completion of the test for the additional 5 mL blood draws. Instruct the subject to not partake in further exercise until completion of the 3rd post exercise blood draw at 24 h. 
		NOTE: These were the time points chosen for this study to evaluate the time line of changes in immune cells. The questions and parameters of interest will dictate when sampling should occur.</li>
	</ol>
	</li>
	<li>Endurance steady-state submaximal cycling test &#8212; Visit 2
	<ol>
		<li>Complete steps 1 for calibration, 3.1 for ECG preparation, 3.2.1 for bike fitting and 3.2.2 for forehead pulse oximeter installation.</li>
		<li>Review the exercise test procedure with the subject. Inform the subject that they will be biking for 45 min, but unlike the maximal exercise test, they will only be asked to breathe through the mouthpiece with the nose clip for sections of test and not continuously. 
		NOTE: As with the maximal exercise test, HR and rhythm will be continuously monitored by the 12-lead ECG. SpO2 will be continuously monitored with the pulse oximeter.</li>
		<li>Draw 5 mL of venous blood sample from the antecubital vein (baseline endurance submaximal exercise blood draw) before starting the test.</li>
		<li>Instruct the subject to grasp the mouthpiece by mouth and attach the nose clip.</li>
		<li>Begin data collection, and then start the exercise protocol. Instruct the participant to start pedaling, and ask them to reach a pedal rate between 60 and 80 rpms. 
		NOTE: For this study the endurance steady-state protocol is 45 min with a 3&#8211;5 min warm up at 50 W. Following warmup, the wattage is increased to 60% of the subject&#8217;s maximal workload determined from visit 1. This is different from visit 1 in that the workload is constant and the bout is set for a specific duration, rather than increasing the workload until peak VO2 is reached.</li>
		<li>Measure BP (use the same technician as before) and ask the subject to report exertion level intermittently (every 3&#8211;5 min) throughout the exercise.</li>
		<li>Instruct the subject to release the mouthpiece from the 10th min to 25th min, and re-grasp the mouthpiece from the 25th min to 30th min and during last 5 min of 45 min bout (40th min to 45th min). 
		NOTE: Gas exchange metrics are being intermittently monitored only when the subject is on the mouthpiece; intermittent monitoring is done as the mouthpiece can be dry and uncomfortable for the subject when used for longer periods of time. Since the goal of this type of exercise test is to have the subject reach specified exercise intensity and then hold that as a steady-state, the subject&#8217;s gas exchange does not need to be continuously monitored unless it is a primary outcome measure. In the case of this study it was the stimulus, and not the outcome of interest.</li>
		<li>Ensure that the test remains at a steady-state by monitoring the following metrics:
		<ol>
			<li>Check that the VO2 hasn&#8217;t increased significantly (+/- 5 mL/min/kg) when the subject is back on the mouthpiece.</li>
			<li>Check that the subject&#8217;s HR doesn&#8217;t increase by more than 5 bpm.</li>
			<li>Observe for the subject appearing tired or that his/her RPE rating is rising.</li>
		</ol>
		</li>
		<li>Drop the workload by ~5&#8211;10% to ensure completion of 45 min of cycling, if any of the above occurs.</li>
		<li>Following 45 min, instruct the participant to complete a 2 min recovery period of easy pedaling, and then draw 5 mL of venous blood (post exercise blood draw).</li>
		<li>Give the same post exercise instructions as in visit 1 i.e., do not engage in exercise until after the 24 h time point and return to the lab for blood draws 3 h and 24 h post exercise.</li>
	</ol>
	</li>
</ol>
4. Blood Analysis
<ol>
	<li>Process blood samples for analysis. 
	NOTE: Potential methods may include, but are not limited to, flow cytometry of circulating leukocytes, cytokine analysis of plasma samples, and/or leukocyte gene expression analysis. Additionally, optimal time points may need to be empirically determined.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Power:+An+Important+Fatigue+Threshold+in+Exercise+Physiology.">Critical Power: An Important Fatigue Threshold in Exercise Physiology.</a> Medicine and Science in Sports and Exercise. 48 (11), 2320-2334 (2016).</li><li>Gustafsson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Acute+Exercise+on+Circulating+Soluble+Form+of+the+Urokinase+Receptor+in+Patients+With+Major+Depressive+Disorder.">Effects of Acute Exercise on Circulating Soluble Form of the Urokinase Receptor in Patients With Major Depressive Disorder.</a> Biomarker Insights. 12, 1177271917704193 (2017).</li><li>Hallberg, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+release+of+cytokines+in+patients+with+major+depressive+disorder.">Exercise-induced release of cytokines in patients with major depressive disorder.</a> Journal of Affective Disorders. 126 (1-2), 262-267 (2010).</li><li>Bengtsson Lindberg, M., Wilke, L., Vestberg, S., Jacobsson, H., Wis&#233;n, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+Release+of+Cytokines/Myokines+in+a+Single+Exercise+Test+before+and+after+a+Training+Intervention+in+Patients+with+Mild+Cognitive+Impairment.">Exercise-induced Release of Cytokines/Myokines in a Single Exercise Test before and after a Training Intervention in Patients with Mild Cognitive Impairment.</a> International Journal of Physical Therapy &amp; Rehabilitation. 3, (2017).</li></ol>

The authors declare that they have no competing financial interests.

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 &#34;dose&#34; 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 tailo...

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 &#34;medicine&#34; 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&#39;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 &#34;overbuilt,&#34; 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,&#160;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&#160;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.

<table><tbody><tr><td>Metabolic cart/portable system</td><td>MCG Diagnostics</td><td>Mobile Ultima CPX System</td><td>The flow calibration syringe, and calibration gases should come with system. There are numerous possible options/alternatives.</td></tr><tr><td>Pulmonary function software (Breeze Suite)</td><td>MCG Diagnostics</td><td></td><td>Software used will depend on the metabolic system</td></tr><tr><td>Upright cycle ergometer</td><td>Lode ergoline</td><td>960900</td><td>Numerous possible options/alternatives</td></tr><tr><td>12-Lead ECG</td><td>GE Healthcare</td><td>CASE Exercise Testing System</td><td>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</td></tr><tr><td>Pulse oximeter</td><td>Masimo</td><td>MAS-9500</td><td>Usually multiple probe options: finger, forehead, ear lobe.&amp;nbsp; Usually avoid finger as tight handlebar grip can cause measurement inaccuracies</td></tr><tr><td>Pneumotach (preVent Flow Sensor)</td><td>MCG Diagnostics</td><td>758100-003</td><td>Alternative systems can use a turbine</td></tr><tr><td>Nose piece (disposable)</td><td>MCG Diagnostics</td><td>536007-001&amp;nbsp;</td><td>Numerous possible options/alternatives</td></tr><tr><td>Mouthpeice with saliva trap</td><td>MCG Diagnostics</td><td>758301-001</td><td>Suggest filling the saliva trap with paper towel/gauze and tape cap to limit dripping</td></tr><tr><td>Headband</td><td>Cardinal Health</td><td>292866</td><td>Used to secure the forehead pulse oximeter and the lines for the cart</td></tr><tr><td>Stethescope&amp;nbsp;</td><td>3M Littman</td><td>3157SM</td><td>Numerous possible options/alternatives</td></tr><tr><td>Blood pressure cuff</td><td>HCS</td><td>HCS9005-7</td><td>Cuff size will depend on the population planning to test</td></tr><tr><td>ECG Electrodes</td><td>Cardinal Health</td><td>M2570</td><td>only needed with lead based ECG/HR monitoring</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;EDTA tube 5 mL</td><td>Becton Dickinson</td><td>368661</td><td></td></tr><tr><td>*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.</td><td></td><td></td><td></td></tr></tbody></table>

conducting maximal and submaximal endurance exercise testing to measure physiological and biological responses to acute exercise in humans

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.

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 respect...

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Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

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 ...

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20.1K Views.  Mayo Clinic. 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.

Video: Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Behavior

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen consumption is typically used as the criterion measurement in this regard, two metabolic breakpoints that reflect changes in the dynamics of lactate production/consumption as the work rate is increased are perhaps more relevant for endurance athletes from a functional standpoint. Exercise economy, which represents the rate of oxygen consumption relative to performance of submaximal work, is also an important parameter to measure for endurance-athlete assessment. Ramp incremental tests comprising a gradual but rapid increase in work rate until the limit of exercise tolerance is reached are useful for determining these parameters. This type of test is typically performed on a cycle ergometer or treadmill because there is a need for precision with respect to work-rate incrementation. However, athletes should be tested while performing the mode of exercise required for their sport. Consequently, swimmers are typically assessed during free-swimming incremental tests where such precision is difficult to achieve. We have recently suggested that stationary swimming against a load that is progressively increased (incremental tethered swimming) can serve as a &#34;swim ergometer&#34; by allowing sufficient precision to accommodate a gradual but rapid loading pattern that reveals the aforementioned metabolic breakpoints and exercise economy. However, the degree to which the peak rate of oxygen consumption achieved during such a protocol approximates the maximal rate that is measured during free swimming remains to be determined. In the present article, we explain how this rapidly incremented tethered-swimming protocol can be employed to assess the cardiorespiratory capacity of a swimmer. Specifically, we explain how assessment of a short-distance competitive swimmer using this protocol revealed that his rate of oxygen uptake was 30.3 and 34.8 mL&#8729;min-1&#8729;kg-1BM at his gas-exchange threshold and respiratory compensation point, respectively.

As opposed to measurement during free swimming, which presents inherent challenges and limitations, determination of important parameters of cardiorespiratory function for swimmers can be made using a more feasible and easier to administer tethered-swimming rapidly incremented protocol with gas exchange and ventilatory data collection.

Incremental exercise testing is the standard means of assessing cardiorespiratory capacity of endurance athletes. While the maximal rate of oxygen ...

Biochemistry

Evaluation of Exercise Tolerance of Pigs Subjected to Exercise

Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based therapeutic angiogenesis is a promising approach for treating patients with significant symptoms, despite optimal pharmacological therapy and invasive procedures. However, many promising cardiovascular gene therapy techniques have failed to accomplish expectations in clinical trials. One explanation is a mismatch between preclinical and clinical endpoints used to measure efficacy. In animal models, the emphasis has usually been on easily quantifiable endpoints, such as the number and area of the capillary vessels calculated from histological sections. Apart from mortality and morbidity, endpoints in clinical trials are subjective, such as exercise tolerance and quality of life. However, the preclinical and clinical endpoints likely measure different aspects of the applied therapy. Nevertheless, both types of endpoints are required to develop successful therapeutic approaches. In clinics, the main goal is always to alleviate patients' symptoms and improve their prognosis and quality of life. To achieve better predictive data from preclinical studies, endpoint measurements must be better matched to those in clinical studies. Here, we introduce a protocol for a clinically relevant treadmill exercise test in pigs. This study aims to: (1) provide a reliable exercise test in pigs that can be used to evaluate the safety and functional efficacy of gene therapy and other novel therapies, and (2) better match the endpoints between preclinical and clinical studies.

Author Spotlight: Exercise Test for Evaluation of the Functional Efficacy of the Pig Cardiovascular System  

The present protocol describes a large animal exercise test model to assess the functional capacity of the cardiovascular system for evaluating the efficiency of new therapies in the preclinical setting. It is comparable to a clinical exercise test.

Despite the progress in treatments, cardiovascular diseases are still one of the biggest causes of mortality and morbidity worldwide. Gene therapy-based ...

Genetics

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has been described. The exercise-induced cTnT response distorts the diagnostic role of the cTnT assay. Although high-intensity interval exercise (HIE) is growing in popularity and concerns remain about its safety, available data related to cTnT release after HIE is limited, which hampers the use of HIE as a health intervention. Here, we present three representative HIE protocols [traditional HIE (repeated 4 min cycling at 90% V&#775;O2max interspersed with 3 min rest, 200 kJ/session); sprint interval exercise (SIE, repeated 1 min cycling at 120% V&#775;O2max interspersed with 1.5 min rest, 200 kJ/session); and repeated sprint exercise (RSE, 40 x 6 s all-out sprints interspersed with 9 s rest)] and one representative MCE protocol (continuous cycling exercise at an intensity of 60% V&#775;O2max, 200 kJ/session). Forty-seven sedentary, overweight young women were randomly assigned to one of four groups (HIE, SIE, RSE, and MCE). Six bouts of respective exercise were performed by every single group, with each being 48 h apart. Meanwhile, for four groups, the duration of the entire testing period was identical, being 10 days. Before and after the first and final exercise bouts, an assessment was conducted of cTnT. The current study provides a frame of reference giving a clear picture of how a specific exercise session affects the circulating cTnT concentration at the early stage of training. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise, especially for HIE.

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has ...

Determining The Electromyographic Fatigue Threshold Following a Single Visit Exercise Test

Theoretically, the electromyographic (EMG) fatigue threshold is the exercise intensity an individual can maintain indefinitely without the need to recruit more motor units which is associated with an increase in the EMG amplitude. Although different protocols have been used to estimate the EMG fatigue threshold they require multiple visits which are impractical for a clinical setting. Here, we present a protocol for estimating the EMG fatigue threshold for cycle ergometry which requires a single visit. This protocol is simple, convenient, and completed within 15-20 min, therefore, has the potential to be translated into a tool that clinicians can use in exercise prescription.

This protocol describes the electromyographic fatigue threshold which demarcates between nonfatiguing and fatiguing exercise workloads. This information could be used to develop a more individualized training program.

Theoretically, the electromyographic (EMG) fatigue threshold is the exercise intensity an individual can maintain indefinitely without the need to recruit ...

Experimental Protocol of a Three-minute, All-out Arm Crank Exercise Test in Spinal-cord Injured and Able-bodied Individuals

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be small; therefore, sensitive tools are fundamental to exercise physiology. There are currently many exercise tests that allow for the examination of exercise capacity in able-bodied athletes, with protocols mainly for lower-body or whole-body exercise. There is a trend to test athletes in a sport-specific setting that closely resembles the actions that the participants are used to performing. Only a few protocols test short-term, high-intensity exercise capacity in participants with an impairment of the lower body. Most of these protocols are very sport-specific and are not applicable to a wide range of athletes. One well-known test protocol is the 30 s Wingate test, which is well-established in cycling and in arm crank exercise testing. This test analyzes high-intensity exercise performance over a 30 s time duration. In order to monitor exercise performance over a longer duration, a different method was modified for application to the upper body. The 3 min, all-out arm crank ergometer test allows athletes to be tested in a manner specific to 1,500 m wheelchair racing (in terms of exercise duration), as well as to upper body exercises such as rowing or hand-cycling. In order to increase the reliability with identical test conditions, it is crucial to precisely replicate settings such as the resistance (i.e., torque factor) and the position of the participants (i.e., the height of the crank, the distance between the crank and the participant, and the fixation of the participant). Another important issue concerns the beginning of the exercise test. Fixed revolutions per minute are required to standardize the test conditions for the start of the exercise test. This exercise protocol shows the importance of accurate operations to reproduce identical test conditions and settings.

We present a protocol to test the aerobic and anaerobic power of the upper body muscles over a duration of 3 min in able-bodied as well as in paraplegic and tetraplegic individuals. The protocol presents specific modifications in its application for upper-body exercise in individuals with or without disability.

Reliable exercise protocols are required to test changes in exercise performance in elite athletes. Performance improvements in these athletes may be ...

Improving Strength, Power, Muscle Aerobic Capacity, and Glucose Tolerance through Short-term Progressive Strength Training Among Elderly People

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in elderly people performing short-term resistance training (RET). Supervised progressive resistance training for 1 h three times a week over 8 weeks was performed by RET participants (71&#177;1 years, range 65-80). Compared to a control group without training, the RET showed improvements on the measures used to indicate strength, power, glucose tolerance, and several parameters of muscle aerobic capacity. Strength training was performed in a gym with only robust fitness equipment. An isokinetic dynamometer for knee extensor strength permitted the measurement of concentric, eccentric, and static strength, which increased for the RET group (8-12% post- versus pre-test). The power (rate of force development, RFD) at the initial 0-30 ms also showed an increase for the RET group (52%). A glucose tolerance test with frequent blood glucose measurements showed improvements only for the RET group in terms of blood glucose values after 2 h (14%) and the area under the curve (21%). The blood lipid profile also improved (8%). From muscle biopsy samples prepared using histochemistry, the amount of fiber type IIa increased, and a trend towards a decrease in IIx in the RET group reflected a change to a more oxidative profile in terms of fiber composition. Western blot (to determine the protein content related to the signaling for muscle protein synthesis) showed a rise of 69% in both Akt and mTOR in the RET group; this also showed an increase in mitochondrial proteins for OXPHOS complex II and citrate synthase (both ~30%) and for complex IV (90%), in only the RET group. We demonstrate that this type of progressive resistance training offers various improvements (e.g., strength, power, aerobic capacity, glucose tolerance, and plasma lipid profile).

The effect of short-term resistance training on elderly people was investigated through the simultaneous use of several methods. Compared to a control group, many improvements were seen, including on muscle aerobic capacity, glucose tolerance, strength, power, and muscle quality (i.e., protein involved in cell signaling and muscle fiber type composition).

This protocol describes the simultaneous use of a broad span of methods to examine muscle aerobic capacity, glucose tolerance, strength, and power in ...

Lower Limb Biomechanical Analysis of Healthy Participants

Biomechanical analysis techniques are useful in the study of human movement. The aim of this study was to introduce a technique for the lower limb biomechanical assessment in healthy participants using commercially available systems. Separate protocols were introduced for the gait analysis and muscle strength testing systems. To ensure maximum accuracy for gait assessment, attention should be given to the marker placements and self-paced treadmill acclimatization time. Similarly, participant positioning, a practice trial, and verbal encouragement are three critical stages in muscle strength testing. The current evidence suggests that the methodology outlined in this article may be effective for the assessment of lower limb biomechanics.

This article introduces a comprehensive experimental methodology on two of the latest technologies available to measure the lower limb biomechanics of individuals.

Biomechanical analysis techniques are useful in the study of human movement. The aim of this study was to introduce a technique for the lower limb ...

A Real-World High-Intensity Interval Training Protocol for Cardiorespiratory Fitness Improvement

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, few studies have tested the efficiency of HIIT protocols in a &#34;real world&#34; setting, e.g., HIIT protocols designed for outdoor spaces without specialized equipment. This study presents a &#34;real world&#34; training protocol, named &#34;beep training&#34;, and compares the efficiency of a HIIT regiment versus a traditional long-duration Moderate-Intensity Continuous Training (MICT) regiment using this beep training protocol on VO2 max of overweight untrained men. Twenty-two subjects performed outdoor running with MICT (n = 11) or HIIT (n = 11). Cardiorespiratory fitness was assessed before and after training protocols using a metabolic analyzer. Both training protocols were performed 3 days a week for 8 weeks using the Beep Test results. The MICT group performed the exercise program at 60%-75% of the maximum speed of the 20 m shuttle test (Vmax) and with a progression of the distance of 3,500-5,000 m. The HIIT group performed the interval exercise with 7-10 bouts of 200 m at 85%-100% of the maximum speed of the 20 m shuttle test (Vmax), interspersed with 1 min of passive recovery. Although the HIIT group presented a significantly lower training volume than the MICT group (p &#60; 0.05) after 8 weeks of beep training, HIIT was superior to MICT in improving VO2 max (MICT: ~4.1%; HIIT: ~7.3%; p &#60; 0.05). The &#34;real world&#34; HIIT regiment based on beep training protocol is a time-efficient, low-cost, and easy-to-implement protocol for overweight untrained men.

This study presents a low-cost and easy-to-implement "real world" high-intensity interval training (HIIT) protocol for scientific research and discusses its efficiency for cardiorespiratory fitness.

High-Intensity Interval Training (HIIT) has emerged as an interesting time-efficient approach to increase exercise adherence and improve health. However, ...

Using Near-Infrared Spectroscopy Wearable Devices to Identify Central Versus Peripheral Limitations During Exercise

The gold standard to assess the aerobic capacity in physically active subjects and athletes is the maximal oxygen consumption test (VO2&#8211;max), which involves analysis of exhaled&#8211;gases and cardiorespiratory variables obtained via the breath-by-breath method in an ergospirometer during an incremental exercise. However, this method cannot elucidate metabolic changes at the muscular level. Near-infrared spectroscopy (NIRS) has emerged as a valuable technology to evaluate local oxygen levels (Tissular Saturation Index, TSI) by quantifying the concentrations of oxygenated (O2-Hb) and deoxygenated (H-Hb) hemoglobin in the microvasculature of tissues. NIRS applications extend to respiratory and locomotor muscles, assessing metabolic changes associated with the cost of breathing (COB) and peripheral workload, respectively. Additionally, cerebral regions, such as the prefrontal cortex, have been explored with NIRS technology to assess physiological changes related to cognitive demand associated with planning or ideation of motor tasks linked to sports performance. Thus, by analyzing exercise-induced changes (D) in O2-Hb, H-Hb, and TSI, it is possible to identify central and peripheral exercise limitations, particularly when endurance training is the main component of physical fitness (e.g., running, cycling, triathlon, etc.). Addressing these factors is paramount for coaches and exercise physiologists to optimize athletic performance, incorporating training strategies focused on the primary exercise-limiting factors. This study outlines a protocol for utilizing wearables devices equipped with NIRS technology to analyze exercise changes in TSI, O2-Hb, and H-Hb, alongside cardiorespiratory variables typically registered in athletes during VO2&#8211;max tests. This approach offers a comprehensive method for identifying the primary systems involved in stopping exercise progression and sports performance improvement.

This protocol integrates near-infrared spectroscopy (NIRS) technology to assess localized hematological and oxygenation changes at the prefrontal cortex, respiratory (m.Intercostales), and locomotor (m.Vastus Lateralis) muscles during cardiopulmonary exercise testing, enabling the identification of central and peripheral limiting factors affecting exercise performance.

The gold standard to assess the aerobic capacity in physically active subjects and athletes is the maximal oxygen consumption test (VO2&#8211;max), which ...