Introduction:

VO₂ refers to the volume of oxygen consumed and taken up by working tissues in order to produce energy. VO_{2 Max} refers to the maximum amount of oxygen that an individual can utilize during intense or maximal exercise that could not be increased despite further increases in exercise workload, thereby defining the limits of the cardiorespiratory system (Hill et al., 1923). This measurement is generally considered the best indicator of cardiovascular fitness and aerobic endurance (Cappelli et al., 2019). The more oxygen a person can use during high-intensity exercise, the more energy a person can produce. Being able to consume more oxygen helps athletes sustain a high-intensity workload for a longer period of time. Individuals with a VO_{2 Max} below 18 mL/kg/min in men and 15 mL/kg/min in women become very challenged to autonomously complete activities of daily living.

An increase of 25% in VO_{2 Max} is equivalent to gaining back an estimated 12 years of vigor to one’s lifestyle (Shephard, 2008).

In athletes, increasing this value will lead to improved performance. At high levels of performance however, it is more difficult to improve than in sedentary or moderately active populations. Therefore, many elite endurance athletes will focus on biomechanical efficiency and the economy as to use less energy at a given speed.

So, if an athlete can sustain performance at a greater percentage of their VO_{2 Max} for a longer period of time, they will have an edge over their competition due to their increased power output. At the elite level of competition, even the slightest improvements in one’s performance can make a difference in how well one places. Often, it is a matter of inches and milliseconds that determine a winner. Depending on the nature of the sporting event, elite athletes have a VO_{2 Max} in the range of 70 mL/kg/min to approximately 80mL/kg/min in men, and 60 mL/kg/min to 70 mL/kg/min in women.

Figure 1 in the appendix shows normative ranges for VO_{2 Max} values in males and females.

For instance, cross-country skiers have the highest VO_{2 Max} levels in the world because they train using the greatest degree of muscle mass in an upright position (Cappelli et al., 2019). Endurance runners and cyclists fall just below, as they use slightly less muscle mass in their respective events. Absolute VO₂ is measured in liters per minute, and relative VO₂ is measured as milliliters per kilogram of body weight per minute. Correcting for body mass enables researchers to compare data between individuals of different ages, genders, and fitness levels using a standardized scale.

The Fick equation states:

VO₂ (mL/min) = Cardiac Output (mL/min) * (a-v)O₂ difference (mL/100 mL of blood)

Cardiac output can be broken down into two components: Heart Rate (HR) and Stroke Volume (SV). The Fick equation can then be modified to understand each component and how it contributes to VO₂ values. The revised equation states:

VO₂ (mL/min) = Stroke Volume (mL/beat) * Heart Rate (beats/minute) * (a-v)O₂ difference (mL/100 mL of blood).

The three components of the Fick Equation all relate to how oxygen circulates around the body and the rate at which it is used. Heart Rate and Stroke Volume deal with the delivery of oxygen through the blood as it is ejected from the heart. When determining VO_{2 Max}, HR max is considered, and not a submaximal heart rate. Heart rate max gradually declines with age, as determined by the equation 220 – age = HR max. This makes it impossible to improve as a factor of cardiac output. Therefore, the adaptations to cardiac output seen from cardiovascular training have a greater effect on the stroke volume component of cardiac output. Prolonged aerobic exercise training may also increase stroke volume, which frequently results in a lower (resting) heart rate.

Reduced heart rate prolongs ventricular diastole (filling), increasing end-diastolic volume, and ultimately allowing more blood to be ejected (Betts et al., 2013). In other words, along with an improvement in cardiac output comes an increased stroke volume relative to the changes in heart rate. The third component, arterial-venous oxygen difference, provides information about the body’s peripheral delivery of oxygen to working tissues. This component is important when considering exercise because peripheral delivery of blood is what feeds the active muscles the oxygen necessary to maintain a given exercise intensity. The difference in oxygen concentration in arterial and venous blood signifies how much oxygen is being extracted out of the blood and into the working muscles.

A high (a-v) O₂ difference is desired because that indicates that the muscles are provided with the adequate oxygen to sustain exercise.

There are physiological and anatomical factors that account for gender- and age-specific differences in VO_{2 Max} values. Males tend to perform better absolutely than females on tests of aerobic fitness. Females generally have a greater percentage of body fat and a lower level of muscle mass that may be disadvantageous during this testing. In addition to muscle mass and fat distribution, males have a higher red blood cell count, increasing their hemoglobin and oxygen-carrying capacity. In addition, men tend to have larger ventricular dimensions. These can fill with more blood and contribute to greater stroke volume.

Oxygen consumption, therefore, is reliant on the oxygen-carrying capacity of the blood, the ability of the heart to circulate oxygen-carrying blood (Cardiac Output), and the ability of the peripheral tissues, such as skeletal muscle, to extract and utilize oxygen (a-v O₂ difference). However, the ability to exercise the skeletal muscle to extract and utilize oxygen greatly exceeds the ability of the heart to circulate blood. For this reason, VO_{2 Max} is primarily limited by maximal cardiac output and individual differences in VO_{2 Max} is are directly related to differences in maximal cardiac output. As maximal heart rate is not affected by training, individual differences in maximal cardiac output are a function of differences in maximal stroke volume. Therefore, the limiting factor of VO_{2 Max} is stroke volume.

VO_{2 Max} is usually determined during a treadmill or bicycle ergometer test with progressive intensity leading to exhaustion in 8 to 12 minutes.

Values achieved during cycling are normally 90% of those observed on the treadmill because, theoretically, not enough muscle mass is engaged in the exercise to maximally stress the cardiovascular system. For this reason, values obtained from a cycling protocol are often referred to as VO_{2 Peak} rather than VO_{2 Max} (Cappelli et al, 2019).

In addition to the VO_{2 Max} obtained from a maximal exercise test, additional cardiovascular data is required to further analyze one’s performance. As previously mentioned, cardiac output is a key limiting factor to endurance performance. Cardiac output can be measured directly, using a catheter, which is invasive and could cause a subject discomfort while exercising. Cardiac output can also be measured indirectly with the use of a piece of equipment called a Physioflow, a non-invasive impedance cardiograph device that measures cardiac output. Recommended electrode placements involve six electrodes, all providing various views of the heart to determine the rate, rhythm, and axis of the electrical activity of the heart.

The Physioflow measures cardiac output based on Thoracic Electrical Bioimpedance (TEB). TEB changes with the amount of blood in the thoracic artery system. The more blood the heart is pumping, the greater the conductivity of the thorax. This is because blood is a liquid, and liquids are good conductors of electrical signals. There is another potential method of non-invasively estimating stroke volume. This is done by measuring oxygen pulse, the amount of oxygen (mL) consumed with each beat of the heart, commonly expressed as mL O₂ per beat. This is directly proportional to SV, expressed as mL of blood per beat of the heart.

Oxygen pulse is provided during graded exercise testing on the metabolic cart and can be calculated as follows:

O₂ Pulse (mL O₂/beat) = VO₂ (mL O₂/min) / Heart Rate (beats/minute).

Cardiac output is commonly identified as one of the main limiting factors to oxygen delivery and VO2max (Bassett & Howley 2000). In fact, some researchers have concluded that 70-85% of the limitation in VO2max can be attributed to maximal cardiac output (Cerretelli & DiPrampero, 1987). A person’s maximal heart rate is quite stable and remains unchanged with endurance training. Maximal heart rate is much more dependent on a person’s age, decreasing as one age. On the other hand, stroke volume increases substantially from endurance training. Much of this rise is due primarily to the increased chamber size and the wall thickness of the left ventricle (Kravitz & Dalleck, 2002). However, from endurance training, both the left and right ventricles have expanded capacity to fill with blood.

The heart, being a muscle with the ability to extend, also attains a greater stretch from the increased blood volume. This results in a stronger elastic recoil for ejecting the blood to the body tissues. In other words, the concentric and eccentric hypertrophies of the left and right ventricles lead to a subsequent increase in ejection fraction (Betts et al., 2013). Ejection fraction is a measurement, expressed as a percentage, of how much blood the left ventricle pumps out with each contraction. An ejection fraction of 60 percent means that 60 percent of the total amount of blood in the left ventricle is pushed out with each heartbeat. The normal range for ejection fraction is 50 to 70 percent.

The variation in individual maximal stroke volume explains most of the range observed in VO2max in trained and untrained individuals.

During incremental exercise to maximal, untrained individuals experience a plateau in stroke volume at an intensity approximately 50% VO2max, whereas with highly trained endurance athletes, stroke volume continues to increase up to VO2max (Robergs & Roberts 2000). This allows for further increases in cardiac output and improved endurance performance.

VO₂ has been shown to be a strong and independent predictor of all-cause and disease-specific mortality (McKinney et al., 2016). The purpose of this experiment is to identify the strongest limiting factor of VO_{2 Max}. Figure 2 in the appendix shows the relationship between activity/fitness level and risk for disease/mortality. By analyzing the VO₂ values from this experiment and considering each individual component of the Fick equation and how it applies to the changes seen throughout the duration of the exercise test, it is possible to determine which aspects contribute the most to a change in VO_{2 Max} using correlation coefficients and linear regression models.

Manipulating variables of an exercise protocol to challenge the cardiovascular system will lead to adaptations such as ventricular hypertrophy, ultimately strengthening the function of the heart (Betts et al., 2013). Therefore, in theory, increasing one’s VO_{2 Max} through a prescribed exercise intervention targeted at improving cardiovascular function can serve as a protective mechanism against disease.

Methods and Materials:

The subjects of the study (N=2) were one male and one female. Subject 1 was instructed to perform a VO_{2 Peak} test on an electronically braked Monark 828E ergometer, and Subject 2 was instructed to perform a VO_{2 Max} test on a Cardinal Health Trackmaster treadmill. Prior to beginning the test, the metabolic cart used to collect oxygen consumption (VO₂) and carbon dioxide production (VCO₂) data was calibrated.

A metabolic cart can also be used to assess the energy requirements during exercise and to determine work capacity. The cylinder used to calibrate the cart contains a known volume (3 liters) of air and is used as a reference point to compare the volume of gas breathed by the subject throughout the test. This ensures accurate VO₂ and VCO₂ measurements when running the test. The hose from the cylinder was connected to the flow sensor and using a series of slow and light plunges to faster and harder plunges, the technician calibrating the cart was instructed to keep the line within ranges on the graph on the screen.

The gas calibration was then done using two gas tanks, one containing oxygen and the other containing carbon dioxide in the same concentrations as the atmosphere (approximately 21% oxygen and 0.05% carbon dioxide) and compared to the relative volumes of expired air from the test subject (approximately 16% oxygen and 4% carbon dioxide). The calibration was performed by loosening the caps on the gas tanks in the back of the cart and inserting the sample line from the flow sensor into the gas calibration machine.

Data from these experiments were recorded and analyzed in Microsoft Excel using linear regression equations.

Both tests used intervals of 3 minutes between stages. This is enough time to allow the subject to enter a steady-state heart rate. Steady-state is the period in which the heart rate stays relatively constant throughout a given stage of exercise (within 6 bpm from the beginning of the stage to the end of the stage). An HR max was established for each subject using the formula: 220 – age = HR max. For subject 1, HR max is 198, and for subject 2, HR max is 198.

Subject 1 is a 22-year-old female who is 172 cm tall and weighs 66 kg. In addition to the ergometer, other materials used for testing included a Polar T31C heart rate monitor, a metabolic cart, the Physioflow, and a clipboard for the subject to indicate her RPE. The subject was properly fitted to the Monark bike ergometer. She was correctly fitted with a metabolic mask and head harness. The mask air seal was examined to ensure there was no air being leaked out during the test.

The subject was fitted with the six leads of the Physioflow:

1. The probe sites should be shaved, with a disposable surgical razor if necessary.
2. Then, these sites should be cleaned off with an alcohol prep pad and dried with a paper towel. Rub the skin with Nuprep abrasive gel using gauze, removing the excess gel when done.
3. Connect the matching colored Physioflow probes on the patient cable to the Physioflow PF50 AgCl electrodes.
4. Correctly place the six electrodes onto subject one at the proper locations. The electrode locations are as follows: Z1 (white) superior to Z2 (blue) on the left carotid artery, EKG1 (red) slightly to the right of the sternum, Z3 (green) on the back at the same level of the xiphoid process of the sternum, Z4 + EKG3 (black) right below Z3 on the back, and EKG2 (yellow) at the left anterior axillary line.

Blood pressure was taken with the subject in exercise position and the Physioflow was calibrated using this blood pressure measure. A resting, baseline metabolic measurement was taken for 2 minutes with the subject seated and resting on the ergometer. The subject warmed up by pedaling at 50 rpm on an unloaded Monark bike for three minutes. During this time, heart rate, ventilation, VO₂, VCO₂, cardiac output, and stroke volume were observed. Following the warm-up stage, the workload was increased to 50 watts (300 kpm/min) for 3 minutes. Values were recorded 15 seconds before the end of the stage. The workload was increased by 25 watts every 3 minutes for 6 total stages until the subject reached exhaustion after 175 watts.

The subject was then instructed to do a cool-down to allow for recovery of pre-exercise heart rate and blood pressure.

Subject 2 is a 22-year-old male who is 172.7 cm tall and weighs 68 kg. In addition to the ergometer, other materials used for testing included a Polar T31C heart rate monitor, a metabolic cart, and a clipboard for the subject to indicate her RPE. He was correctly fitted with a metabolic mask and head harness. The mask air seal was examined to ensure there was no air being leaked out during the test. The subject’s resting heart rate was collected prior to stepping on the treadmill. The subject warmed up on the treadmill by walking at 1.7 mph on a 10% incline for 3 minutes. During minutes 2 to 3, heart rate, ventilation, VO₂, and VCO₂ were observed. The intensity was increased based on the subject’s heart rate and RPE every 3 minutes until exhaustion.

The Modified Bruce Protocol Procedure was followed, recording values approximately every 15 seconds before the end of each stage:

Speed (mph)	Incline (%)
1.7	10
2.5	12
3.4	14
4.6	15
5.2	15
5.8	15
6.4	15
7.0	15

The following equations were used to gather and estimate values for (a-v)O₂ difference, cardiac output, stroke volume, and VO₂:

VO₂ (mL/min) = [Cardiac Output (mL/min) * a-v O₂ difference (mL/100 mL blood)]

(a-v)O₂ difference (mL/100 mL blood) = 5.72 + 0.105 * %VO_{2 Max}

(expressed as a whole number percentage: i.e., 75% and not 0.75)

Cardiac Output (mL/min) = [VO₂ (mL/min) / (a-v)O₂ difference (mL/100 mL blood)]

Cardiac Output (mL/min) = Heart Rate (bpm) * Stroke Volume (mL/beat)

Stroke Volume (mL/beat) = Cardiac Output (mL/min) / Heart Rate (bpm)

There are three post-test criteria to determine whether a true max was reached:

1. 1. A plateau in VO₂ despite further increases in workload
   2. A heart rate within 10 beats per minute of the age-predicted maximal heart rate
   3. An R-Value greater than 1.10

Results:

Cycle Ergometer VO_{2 Peak} Test Data:

Stage	VO2 (L/min)	VO2 (mL/kg/min)	VCO2 (L/min)	Power (Watts)	CO (L/min)	HR (bpm)	SV (mL/beat)	(a-v) O2 difference	RPE	CO (L/min)	HR (bpm)	SV (mL/beat)
Warmup	0.44	6.6	0.36	0	6.304	91	69.274	6.98	–	8	112	71.4
1	1.73	26	1.17	50	16.078	124	129.661	10.76	10	8.2	107	76.9
2	1.81	27.2	1.47	75	16.5	143	115.385	10.97	11	10.1	135	75.1
3	1.7	25.6	1.59	100	15.955	162	98.488	10.66	12	13.2	149	88.8
4	2.05	30.9	2.04	125	17.514	178	98.393	11.71	13	17.6	167	105.3
5	2.63	39.6	2.62	150	19.649	189	103.963	13.39	14	17.7	177	99.6
6	2.92	44	3.3	175	20.527	197	104.198	14.23	17	20.3	194	104.4

Treadmill VO_{2 Max} Test Data:

Stage	VO2 (L/min)	VO2 (mL/kg/min)	VCO2 (L/min)	Speed (mph)	Incline (%)	Cardiac Output (L/min)	HR (bpm)	Stroke Volume (mL/beat)	(a-v) O2 difference
1	0.234	3.4	0.188	1.7	10	3.545	123	28.82	6.6
2	1.313	19.3	0.995	2.5	12	12.396	145	85.49	10.59
3	1.994	29.2	1.767	3.4	14	15.198	182	83.51	13.12
4	2.814	41.3	3.267	4.6	15	17.413	202	86.2	16.16

Discussion/Conclusion:

The purpose of conducting maximal tests on the cycle ergometer and treadmill was to identify which component of the Fick equation was the strongest limitation to VO_{2 Max}. Using linear regression models and correlation coefficients to analyze the data provided by the metabolic cart and Physioflow, it was shown to what degree each component of the equation varied with changes in intensity. The results of the experiment aligned with the current scientific literature on disease prevention by means of cardiovascular training, indicating that cardiac output, and more specifically, stroke volume is the greatest limiting factor to VO_{2 Max} (McKinney et al., 2016). Cerretelli et al. have concluded in their research that 70-85% of the limitation in VO2max can be attributed to maximal cardiac output (1987).

For both the cycle ergometer and treadmill tests, it is established that the heart rate increases almost linearly as exercise intensity increases up to a certain point before reaching a maximum value. The age-predicted heart rate also decreases linearly, as this is an effect of aging. Maximal heart rate cannot be increased as one age, and therefore is a more stable component of cardiac output. From this information, it can be concluded that the heart rate is not the limiting factor of VO_{2 Max}. The (a-v) O2 difference is the least variable component of the Fick equation because it has the highest correlation to increases in exercise intensity (R² = 0.99). This indicates that as exercise intensity increases, it is inevitable that the (a-v) O2 difference will thereby increase nearly linearly.

It can be concluded that (a-v) O2 difference cannot be manipulated to improve VO_{2 Max}.

Stroke volume has the greatest variability when compared to exercise intensity. The low R² value (0.63) indicates that there is greater room for improvement in the stroke volume in order to increase VO_{2 Max}. According to previous research, it is concluded that physiological adaptations that result from cardiovascular training specifically target the mechanism by which the heart delivers oxygen- and nutrient-rich blood to the working tissues (Kravitz & Dalleck, 2002). Being able to identify the greatest limitation to improving cardiovascular function enables exercise physiologists to design and implement exercise protocols to reduce a subject’s risk against disease-specific and all-cause mortality (McKinney et al., 2016).

The VO_{2 Max} value from the experiment for subject 1 was 54.2 mL/kg/min, which is considered excellent for females between the ages 20-24. For subject 2, the VO_{2 Max} value was 41.5 mL/kg/min, which is considered fair for males between the ages of 20-24. These ranges are compared to the ACSM’s reference for normative values of VO_{2 Max} provided below in the appendix.

According to the 3 criteria for reaching a VO_{2 Max}, the results indicate that each subject reached exhaustion at their respective maximum intensities. According to the data provided by the metabolic cart, subject 1 reached a plateau in her VO₂ for roughly 2 minutes and 30 seconds where she fluctuated between 44 mL/kg/min and 46 mL/kg/min, despite further increases in intensity. Subject 1’s heart rate elevated to 197 bpm, which falls within 10 bpm of her age-predicted maximal heart rate (198 bpm). The data from the metabolic cart also reveals that subject 1 maintained an R-value above 1.10, fluctuating between 1.11 and 1.16 for approximately 2 minutes and 40 seconds.

Subject 1’s RPE at the final stage of the test was at 17, a high score indicating a very hard perceived workload.

Aaccording to the data provided by the metabolic cart, subject 2 reached a plateau in his VO₂ for approximately 2 minutes, where he fluctuated between 41.1 and 41.3 mL/kg/min, despite further increases in intensity. Subject 2’s heart rate elevated to 202 bpm, which is greater than his age-predicted maximal heart rate (198 bpm). According to the metabolic cart data, subject 2 maintained an R-value above 1.10 from 8^th minute of the test to nearly the 13^th minute. The R-values ranged from 1.11 prior to the final stage to 1.61 when the subject reached volitional failure and entered a subsequent forced recovery and cool-down. Subject 2’s RPE at the 4^th stage of the test was at 20, indicating maximum exertion before the 8^th and final stage was reached. Because each subject reached exhaustion and their maximum VO₂, the validity and reliability of the maximal testing procedure was verified.

The following data is listed in the table titled “Cycle Ergometer VO_{2 Peak} Test Data.”

When comparing the results between Subject 1’s data from the Physioflow (experimental) and the estimations calculated by data from the metabolic cart (estimated), there were some slight differences between the values for stroke volume and cardiac output at several stages throughout the test, however, the beginning and end of the test show very similar values. The beginning values for stroke volume were within 1.9 mL of each other (Estimated: 69.3 mL/beat and Experimental: 71.4 mL/beat). The end values for stroke volume were within 0.2 mL of each other (Estimated: 104.2 mL/beat and Experimental: 104.4 mL/beat).

The calculated stroke volume shows a significant spike up to 129 mL/beat before decreasing and stabilizing near the end value, whereas the Physioflow shows a more gradual and stable set of values throughout the entire duration of the test. The beginning values for cardiac output were within 1.7 L of each other (Estimated: 6.3 L/min and Experimental: 8.0 L/min). The end values for cardiac output were within 0.2 mL of each other (Estimated: 20.5 L/min and Experimental: 20.3 L/min).

The calculations overestimate the values at submaximal intensities but at maximal intensity are 99% of the experimental values given by the Physioflow. There is a 1% margin of error when using the metabolic cart compared to the Physioflow to estimate stroke volume and cardiac output values.

The experiment had some limitations.

For instance, a sample size of 2 subjects makes it difficult to generalize findings towards a larger group of individuals. Having a larger sample size who vary in age, race, gender, etc. will produce results that can be applied to a broader population. Lack of experience exercising with the metabolic mask can contribute to discomfort and difficulty breathing. The mask does not entirely simulate the conditions of running without a mask due to the apparatus having a fixed port through which a subject breathes, limiting the volume of air that can pass through and feel natural. Other contributions to performance can vary between tests such as the subjects’ hydration status, fuel availability, and movement efficiency. As mentioned before, the estimation of values for stroke volume and cardiac output have a greater discrepancy in submaximal values compared to the experimental method using the Physioflow.

Nevertheless, the results from the experiment can be beneficial in taking the next step towards exercise prescribed disease prevention. A relevant potential long-term study would be to design and prescribe an exercise protocol that will yield the anatomical, physiological, and metabolic adaptations necessary to achieve an improved cardiovascular system. Consequently, by observing and analyzing the improvements in stroke volume over time, even as heart rate decreases from aging, VO_{2 Max} should continue to improve with training. Using several methods of screening, a physician or clinical practitioner can determine a person’s risk factors for cardiovascular and metabolic diseases and conclude whether each subject has enhanced their defense or reduced their overall risk by means of VO_{2 Max} improvement.

Appendix

Figure 1: Normal Ranges of VO_{2 Max} Values for Men and Women According to the ACSM

Figure 2: Activity Level vs. Risk for Chronic Disease and Premature Mortality from McKinney et al., 2016.

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