THE POTENTIAL OF PH AS A DETERMINANT OF 
MUSCLE FATIGUE DURING STEADY-STATE EXERCISE 
 
 
 
 
 
 
 
 
by 
ADAM S. ROSENCRANS 
 
 
 
 
 
 
 
 
 
 
A THESIS 
 
Presented to the Department of Human Physiology  
and the Robert D. Clark Honors College  
in partial fulfillment of the requirements for the degree of  
Bachelor of Science 
 
August 2016 
 
An Abstract of the Thesis of 
Adam Rosencrans for the degree of Bachelor of Science 
in the Department of Human Physiology to be taken August 2016 
Title: The Potential of pH as a Determinant of Muscle Fatigue During Steady-State 
Exercise 
Approved~ AA__ 
John Halliwill 
This paper attempts to answer four fundamental questions: what are the 
causes of muscle fatigue during steady state exercise, how is pH related to muscle 
fatigue, what technologies exist to measure pH during exercise, and what future 
steps must be taken to make use of this connection. This paper examines muscle 
fatigue as a whole, as well as the role pH plays in predicting the onset of muscle 
fatigue in exercising muscle. Current literature on the physiological and temporal 
links of pH to lactate threshold and muscle fatigue are examined. This paper makes 
the assertion that while acidosis may not cause fatigue or even be exactly 
temporally correlated with muscular fatigue, there is a strong enough correlation 
between the two for pH measurement to have potential use in preventing muscle 
fatigue and subject dropout during steady state exercise. Finally, there is a review 
of current technology and methods for measuring pH in vivo in order to determine 
the most efficient and practical way forward for pH measurement to be used in this 
manner. 
ii 
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Acknowledgements 
 I would like to thank Professor John Halliwill, Professor Samantha 
Hopkins, and Matthew Ely for being incredibly supportive while helping to guide me 
through the process of creating this thesis. I very much appreciate Professor Hopkins’ 
willingness to provide a unique perspective, as it is often the case that interdisciplinary 
collaboration leads to the creation of new ideas. A big thank you is due to Matthew Ely, 
for being one of the first to bring me into the Halliwill lab, and for always challenging 
me to be better. I would also like to thank the other undergraduate and graduate level 
members of the Halliwill lab for creating an environment of experiential learning. And 
of course I would like to thank John Halliwill for providing me with the opportunity to 
participate in research at the collegiate level, an experience which ended up defining my 
undergraduate studies and providing me with inspiration for the future.  
 I would further like to thank Miriam Jordan, for her patience and 
enthusiasm in helping me navigate the thesis process. Thank you to the fantastic staff of 
the Human Physiology department and the Clark Honors College. Thank you to the 
Giustina family for helping to ensure my studies were possible financially. And finally, 
thank you to my sister Sarah for being a continuous positive influence on my life, and to 
my parents Mary and Greg for making this thesis possible through every single loving 
and supportive action since the day I was born.   
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Table of Contents 
List of Figures v 
Introduction 1 
Review of terminology 4 Glycolysis 4 Acidosis 6 Muscle Physiology 7 Steady-State Exercise 8 Lactate Threshold 8 
Muscle Fatigue 11 
The role of pH 17 Intro to pH 17 Old Ideas on pH 18 Modern research on pH causing fatigue 22 Temporal connection 27 
Methods of measuring pH 41 Future directions for research 49 Conclusion 51 
Bibliography 52 
 
 v 
 
List of Figures  
Figure 1: pH and time during one-legged repetitive isometric knee extensions 30 
Figure 2: H+ concentration and time during maximal isometric foot plantar flexion 32 
Figure 3: Individual continuous pH response during one legged knee extensor exercise
 35 
Figure 4: pH and power output during one legged knee extensor exercise 36 
Figure 5: pH, power output, and fatigue during steady state wrist flexion 37 
Figure 6: pH over time during steady state adductor pollicis contraction 39 
Figure 7: Maximal volumetric contraction and hydrogen concentration during steady 
state adductor pollicis contraction. 39 
Figure 8: Comparison between pH measured by NIRS and by traditional invasive 
methods 47 
Figure 9: Proposed H+ threshold measured by NIRS in an exercising individual 48 
 
 
  
 
Introduction 
The Exercise and Environmental Physiology Lab at the University of Oregon, 
directed by Dr. John Halliwill, conducts research into the “hormonal, neural, or 
metabolic factors that are responsible for changes in the cardiovascular system during 
exposure to environmental and physical stresses” (Halliwill, 2016). Physical stresses, 
such as running, encourage the body to adapt so that the stress is easier to handle in the 
future.  Cardiovascular adaptations involve helping bring more oxygen to the exercising 
muscles, whether through improving heart or lung function, improving muscle 
efficiency, or by building new blood vessels. Environmental stresses, such as cold or 
hot weather, or low oxygen situations at high altitude cause many of the same 
adaptations, albeit through different pathways. With this overarching goal in mind, 
current research is focusing on the benefits exercise can provide to an aging population, 
with specific research in signaling pathways for angiogenesis, muscle healing, and 
inflammatory processes. My research is intended to lead to the use of improved 
technology and methodology to more accurately and effectively continue this research.  
Many experimental protocols that are used within our lab involve making 
subjects exercise for extended periods of time, often up to an hour. These exercises 
include dynamic knee extension, cycle ergometry, treadmill running, and other 
protocols. During such an exercise protocol, if the subject becomes too tired to continue 
for the entire duration we must often discard the data. Becoming too tired to maintain 
work at the same level as it was being performed during exercise is termed muscle 
fatigue. It is thought that this muscle fatigue is related to the body switching to new 
forms of breaking down glucose into energy. Even if the subject is able to continue 
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exercising, these new forms of energy production introduce entirely new physiological 
mechanisms which can interfere with data collection, leading to poor data or even faulty 
conclusions. It is in our best interest to make sure that our subjects are able to maintain 
the prescribed work effort for the entirety of the exercise protocol. Current methods of 
preventing subject dropout involve having the subject perform a maximal effort test to 
see how hard they are capable of working, and then picking a work level based on a 
percentage of their maximal work effort, usually around 60% of maximum. While this 
method has proved fairly effective, it is limited in a couple of ways. First, it is a blanket 
rule applied to all subjects, which ignores physiological variation. Second, it relies on 
obtaining an accurate maximum workload for each subject, which can depend on 
subject motivation. Third, the estimation is based on a guess at where anaerobic 
threshold will occur, and because it is only an estimation, it may prove wrong in some 
cases. This paper is intended to determine a more quantitative measurement that can be 
used to prevent muscle fatigue. Further, it specifically examines the role of pH as a 
determinant of muscle fatigue. 
This paper begins with a review of the terminology and physiological concepts 
necessary to discuss the role that pH plays in muscle fatigue. It then goes on to examine 
the possible causes of muscle fatigue, the literature supporting them, and the ability of 
each to be used as a biomarker to determine the onset of muscle fatigue.  Next, there is a 
discussion of the role of pH in muscle fatigue, beginning with past ideas, examining 
current literature, and drawing conclusions on its usefulness for our purposes. There is 
also a more in-depth discussion of the role lactate threshold plays in aligning with 
anaerobic threshold and acidosis. The next section after that is a review of the 
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technologies that exist to measure intramuscular pH, and the paper concludes with 
recommendations for future research. Overall, I argue that while acidosis may not be 
directly causally or temporally related to the onset of muscle fatigue, there is a close 
enough correlation to justify exploring the use of near-infrared spectroscopy as a tool to 
prevent subjects from exercising at a level above their lactate threshold during steady-
state exercise protocols. 
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Review of terminology 
Glycolysis 
In order to understand many of the physiological concepts that will be discussed, 
a basic knowledge of the way in which humans produce and use energy is necessary. 
Energy is mostly stored in the body in the form of ATP, or adenosine triphosphate. This 
molecule is specifically useful because the three phosphate groups are in a high energy 
conformation (Hall 2013). It takes a lot of energy to attach a third phosphate to 
adenosine diphosphate (ADP), and a lot of energy is released when the third phosphate 
group is let go from ATP. In this manner ATP acts as a sort of battery, holding energy 
until it is needed. There are three main ways in which muscles obtain ATP; creatine 
phosphate, anaerobic respiration, and aerobic respiration (Hall 2013). 
Carbohydrates which are ingested are broken down into glucose, and either 
directly turned into ATP or turned into glycogen for storage. Some ATP is attached to 
creatine in the form of phosphocreatine (PCr). Because this can be immediately broken 
down, PCr provides energy for the first 5-10 seconds of exercise, and at maximal 
exercise (Hall 2013). During exercise, your body takes the glucose stored as glycogen 
in muscles and turns the glucose into ATP (adenosine triphosphate), in addition to using 
free glucose within the blood stream. The two methods of doing so are anaerobic and 
aerobic. Both methods begin by turning glucose into pyruvate, which produces a net 2 
ATP (Hall 2013). Anaerobic respirati7on turns pyruvate into lactate through a very 
simple reaction (producing 1-2 H+ in the process), which can then be turned back into 
glucose in the liver. Because of this, glucose is turned to energy very quickly, albeit in 
an inefficient manner (one glucose molecule only makes 2 ATP molecules). This is 
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useful during sprints around 30 seconds in duration (maximal exercise), when your 
body needs energy quickly. 
After this period of time, your body switches to aerobic respiration, which uses 
pyruvate in a series of reactions termed the Krebs cycle. This cycle begins by breaking 
down pyruvate into acetyl-CoA, which then goes through a circular series of processes 
which ultimately produces NADH to be used in the electron transport chain, and carbon 
dioxide as a byproduct, as well as others (Hall 2013). It is important to note that fats and 
lipids can also be used as a source of acetyl-CoA, as well as amino acids to an extent. 
Within the electron transport chain, NADH and FADH2 provide hydrogen ions which, 
through the creation of a chemical gradient, power a system which synthesizes ATP 
(Hall 2013). This process takes much longer than turning glucose into lactate, but it is 
far more efficient (one glucose molecule makes somewhere around 36 ATP molecules). 
Because of this, exercise which demands a more constant supply of energy (steady state 
exercise) tends to use aerobic respiration (Hall 2013). This is important because both 
methods have a different effect on the pH of muscle. 
The PCr system helps immediately synthesize ATP with little effect on H+ 
concentrations. However, after this system is no longer in use, it begins to resynthesize 
PCr. This is hypothesized to have an alkalization effect on the muscle (Robergs et. al 
2004). When the muscle is only using anaerobic respiration, muscular pH was once 
thought to be lowered due to the production of lactic acid, although this is now known 
to be false. When the muscle is only using aerobic respiration, hydrogen ions are both 
produced during the Krebs cycle and used in the ETC (Hall 2013). The overall impacts 
of both systems on muscular pH are still up for debate. In our research, we often attempt 
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to isolate one system of glycolysis or the other in order to control for variables. When 
exercising for longer than a minute at a rate that is too high in energy demand for just 
aerobic respiration, anaerobic respiration will begin to help supplement the required 
energy. Because the presence of anaerobic respiration will somehow impact muscular 
pH, it is thought that the transition into both types of glycolysis is measurable through 
H+ concentrations.  
Acidosis 
It has been widely accepted for much of the past century that lactic acid 
production directly leads to acidosis of the muscle. However, modern research has 
shown that lactic acid is not produced through anaerobic respiration before dissociating 
into lactate and a hydrogen ion. Instead, lactate is a direct product of the reaction 
(Robergs et al. 2004). Whether this lactate is correlated with acidosis is discussed in 
depth later, as arguments have been made supporting lactate production having both 
alkalinizing and acidifying effects on the muscle. Instead, as discussed in a paper by 
Robergs et al., acidosis is thought to stem from a variety of other intramuscular sources. 
The phosphocreatine system, when breaking down into creatine and ATP, is thought to 
be “alkalinizing to the cell, as a proton is consumed in this reaction” (Robergs et al. 
2004). Additionally, whether blood glucose or glycogen is used as a substrate for 
glycolysis matters, as “using glycogen as the source of G6P, as opposed to blood 
glucose, is less acidifying to muscle during intense exercise” (Robergs et al. 2004). 
Still, both sources have an acidifying effect on the muscle. Another source of 
acidification is thought to be ATP hydrolysis, an essential component of muscle 
contraction, due to the production of a hydrogen ion in the reaction (Robergs et al. 
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2004). Finally, NADH production is thought to produce hydrogen ions as a byproduct, 
overall having an acidifying effect (Robergs et al. 2004)  As with most physiological 
concepts, debate surrounds the exact effects of each system on acidosis. For instance, 
Robergs makes the argument that lactate has an alkalizing effect, while Lindinger 
makes the argument that it has an acidifying effect (Lindinger et. al 2005). The exact 
implications of this debate on the role of pH on muscle fatigue will be discussed later. 
Muscle Physiology 
When discussing muscle fatigue, it is important to have an understanding of how 
muscles contract. There are two important terms that will be used throughout this paper, 
excitation-contraction coupling and cross-bridge cycling. Excitation-contraction 
coupling refers to the multiple steps between a neural signal arriving at the muscle and 
the muscle contracting. The first step of this is acetylcholine being released from the 
alpha motor neuron attached to the muscle. This ACh release depolarizes the muscle 
membrane, and this depolarization travels along the membrane into an indent into the 
muscle called a T-tubule (Hall 2013). The depolarization leads to a ryanodine receptor 
channel on the sarcoplasmic reticulum to open. The opening of this channel allows 
calcium release from the SR, where it can then go on to take part in cross-bridge 
cycling. The SR then reuptakes calcium for later release, and extra calcium is released 
out of the muscle cell in order to repolarize the cell membrane (Hall 2013). This process 
is important because the inhibition of this process at any point can lead to muscular 
fatigue. 
Cross-bridge cycling refers to the process in which muscles contract using actin 
and myosin. Specifically, calcium released from the SR in the previous process attaches 
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to troponin C, which allows a myosin head to bond to actin (Hall 2013). Upon bonding, 
the myosin performs a power stroke, pulling on actin. The myosin is then stuck to the 
actin molecule until a new ATP bonds to it. ATP bonding releases the myosin from the 
actin, and hydrolysis of the ATP resets the myosin for the next cross-bridge cycle (Hall 
2013). Like excitation-contraction coupling, interruption at any point of this cycle can 
lead to muscle fatigue.  
Steady-State Exercise 
One term that is used extensively throughout this paper is steady-state exercise. 
There are multiple types of exercise, between maximal and submaximal, continuous and 
interval, isometric and isotonic, etc. Steady-state exercise simply refers to exercise at 
any intensity at which physiological variables—such as heart rate, breathing rate, blood 
metabolite levels, lactate—remain the same. Steady-state exercise could theoretically 
occur at any exercise intensity below anaerobic threshold, and for any length of time. 
Realistically, steady-state exercise tends to be anywhere from about three to five 
minutes to an hour in length (Hall 2013). While steady state exercise could occur at 5% 
of maximum effort, for the purposes of this paper, I use steady-state exercise to describe 
exercise at or near the anaerobic threshold. It is this intensity of exercise that we most 
often have subjects work, and the purpose of this entire paper is to develop a way of 
ensuring subjects remain at that physiological steady-state. 
Lactate Threshold 
In this paper, I use the term lactate threshold to indicate the point at which 
lactate begins to build up, although there are a number of related concepts which must 
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be discussed. When a subject is working hard enough for both types of glycolysis to be 
necessary, lactate begins to build up in the blood plasma, which is termed the lactate 
threshold (Denadai et al. 2005). It has traditionally been thought that the lactate 
threshold correlates to the onset of muscle fatigue. In the past, we have used a maximal 
exercise test to estimate the intensity where a subject’s lactate threshold is, and then 
pick a point below that for steady state exercise. With this research, we hope to use a 
method of continuous monitoring of pH to not only accurately know intramuscular pH 
during exercise, but hopefully determine when a subject crosses the lactate threshold by 
a subsequent sharp decrease in intramuscular pH. This will allow more accurate 
methods of ensuring subjects remain below lactate threshold during steady state 
exercise, and will hopefully improve dropout rates during studies which require 
extended exercise bouts.  
Lactate threshold is closely related to the ideas of Maximal Lactate Steady State 
(MLSS) and Onset of Blood Lactate Accumulation (OBLA). Because of this, it is not 
uncommon to find researchers using these terms interchangeably. However, there are 
specific differences in the terms. MLSS describes the maximum level of exercise at 
which lactate levels remain steady (Denadai et al. 2005). This recognizes the fact that 
muscles are not only involved in lactate production, but lactate clearance. In truth, some 
minimal level of anaerobic respiration is occurring at any workload, even below lactate 
threshold. However, at some point lactate production outpaces lactate clearance. Right 
before this occurs is termed the MLSS. OBLA is a very similar concept. It would make 
sense that right after crossing the MLSS, the excess lactate would enter the blood and 
mark the onset of blood lactate accumulation. Indeed this is often true, especially in 
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whole body exercise. Contraction of multiple large muscle groups can produce large 
amounts of lactate, which would then begin to accumulate in the blood. However, with 
a very small muscle mass such as in a finger, lactate production might outpace lactate 
clearance in the muscle, while the blood experiences no noticeable rise in lactate levels. 
This finger would have crossed the MLSS while the body as a whole would not have 
reached OBLA (Denadai et al. 2005). For simplicity’s sake, I avoid both of these terms 
in favor of the all-encompassing term lactate threshold.  
Additionally, I often imply in this paper, as do other researchers, that lactate 
threshold and anaerobic threshold occur at the exact same time. Again this is not true. 
Anaerobic threshold refers to the moment when exercise is too great for aerobic 
respiration alone, and anaerobic respiration must also help. Lactate threshold is not 
necessarily dependent on anaerobic threshold, as it is simply a measure of the onset of 
lactate build-up in the muscle. While these two events may not occur at the exact same 
time, they usually occur in close proximity to each other. Additionally, it is very 
difficult to tell if a muscle is using anaerobic respiration or not. However, it is relatively 
simple to measure lactate levels in the muscle. For this reason, in this paper I often use 
lactate threshold as an approximation of anaerobic threshold due to its simplicity in 
measurement. There is a more in-depth discussion on the validity of this assumption 
later.  
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Muscle Fatigue 
Muscle fatigue has been highly debated for many years. The general definition 
of muscle fatigue is a lowered ability for the muscle to produce force, whether due to 
clinical conditions or simply exhaustion. There have been a multitude of proposed 
mechanisms of what causes muscle fatigue, each with their own supporting research. 
Fatigue can be split into two main categories; central and peripheral fatigue. In a 1997 
study by Davis and Bailey, the assertion is made that “the unwillingness to generate and 
maintain adequate CNS drive to the working muscle is the most likely explanation of 
fatigue for most people during normal activities” (Davis and Bailey 1997). CNS fatigue 
has been hypothesized to function through serotonin, dopamine, or acetylcholine 
deficiencies. According to Davis, “Good evidence suggests that increases and decreases 
in brain 5-HT activity during prolonged exercise hasten and delay fatigue, respectively” 
(Davis and Bailey 1997. In addition, “several cytokines have been associated with 
reduced exercise tolerance”, and “ammonia in the blood and brain during exercise could 
also negatively effect the CNS function and fatigue” (Davis and Bailey 1997). Davis 
concludes with “clearly fatigue during prolonged exercise is influenced by multiple 
CNS. . .factors” (Davis and Bailey 1997). Unfortunately, it is not this clear. In 2016, 
Contessa et al. question whether central fatigue even exists. “Unlike the directly 
observable and verifiable influence of peripheral factors of muscle fatigue” they state, 
“direct empirical evidence of central fatigue has yet to be revealed” (Contessa et al. 
2015). Contessa et al. continue on to cite multiple research studies on central fatigue, 
and state that only one study even “attempted to take into account the influence of 
peripheral factors on central fatigue” (Contessa et al. 2015). They went on to perform an 
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experiment, which was “able to replicate empirical results from studies of fatiguing 
contractions. . .without requiring the involvement of central factors”(Contessa et al. 
2015). Whether or not central fatigue exists and to what extent is still clearly under 
debate, and therefore does not provide an easily measurable predictive biomarker of 
fatigue, which is what we are searching for. 
The second main category of fatigue—peripheral fatigue—can further be split 
into two categories; substrate fatigue and metabolite fatigue. By definition, substrate 
fatigue is supposedly caused by a lack of enough substrate to produce energy, while 
metabolite fatigue is supposedly caused by the byproducts of exercise somehow 
inhibiting further contraction. The three main substrates involved in substrate fatigue 
are ATP, glycogen, and creatine phosphate. A deficiency in ATP is the most obvious 
cause of fatigue, as ATP is the body’s form of stored energy. Logically, it would make 
sense if muscles have a set amount of ATP available to them that when stores run out 
during exercise, the muscle would no longer be able to contract. Indeed, a lack of ATP 
would lead to muscle fatigue for this reason. However, there is extensive research 
showing that upon reaching muscle fatigue, ATP levels are still sufficient to maintain 
contraction, as running out of ATP would result in rigor mortis (Jennett 2001). This 
suggests that ATP deficiency is most likely not a primary cause of muscle fatigue in 
healthy individuals. Glycogen plays a similar role as ATP in substrate fatigue. Only a 
small amount of ATP is readily available to the muscle in the form of PCr or glucose in 
the blood. The majority of glucose is stored in the muscle in the form of glycogen, so 
that during extended exercise, the muscle has a steady supply of glucose. It would again 
make logical sense that a deficiency in glycogen could interrupt the supply of glucose to 
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the muscle, leading to fatigue. However, as noted by Ortenblad et al. in 2013, “a direct 
cause-and-effect relationship between glycogen and muscle function remains to be 
established”. This study specifically suggests a link between glycogen and SR calcium 
release during excitation-contraction coupling (Ortenblad et al. 2013). While such a link 
may indeed exist, muscle glycogen is not currently a practical, easy to measure 
substance that could allow us to determine onset of muscle fatigue. The final substrate, 
phosphocreatine, is only used within the first approximately 15 seconds of exercise, or 
at very high intensities. While a strong connection between PCr level and muscle 
fatigue may exist, it would only indicate muscle fatigue at maximal exercise. Because 
we are interested in muscle fatigue during long-term steady state exercise, it is unlikely 
that PCr will be useful to us as a biomarker of muscle fatigue.  
Peripheral metabolic fatigue is the subject of the largest amount of fatigue 
research. Evidence seems to suggest that at least a portion of muscle fatigue is due to 
metabolite production, if not the vast majority of it. One important metabolite is 
hydrogen, which is produced during a wide array of metabolic reactions and serves to 
create a more acidic environment. However, as pH will be the primary focus of this 
paper, it will be discussed later.  
Chloride is physiologically very important to muscle contraction. Cl- channels 
are in both the T-tubule and along the membrane of muscle. Cl- influx is involved 
heavily in depolarization of the membrane, helping to signal calcium release from the 
SR. Cairns et al. suggest that “normal [Cl-] protects against excessive fatigue in 
situations in which run-down of the transsarcolemmal K+ gradient occurs”, such as 
during high stimulation frequencies or tetany (Cairns et al. 2004). These results suggest 
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that Cl- may play a role in preventing muscle fatigue, and decreased Cl- could allow for 
additional fatigue to occur. However, the results also seem to apply mostly to situations 
of maximal contraction, so additional research would be necessary before attempting to 
use Cl- levels to predict muscle fatigue during steady state exercise.  
If Cl- plays an important role in protecting against fatigue in situations where 
potassium concentrations are diminished, then it makes sense to look at the role K+ 
plays in muscle fatigue. Even more so than chloride, potassium plays an essential role in 
maintaining polarization of the sarcolemma and T-tubule. During exercise, muscles 
experience a decrease in potassium concentration as it leaves the muscle. According to 
Clausen et al., “this leads to depolarization, loss of excitability and contractile force.” 
(Clausen et al. 2007). While this has been shown before, “little is known about the 
effects of these physiological increases in extracellular K+. . . on contractile endurance” 
(Clausen et al. 2007). In this paper, Clausen et al. examine the role that potassium plays 
in fatigue in rat muscle. They find that “excitation-induced increase in [K+] is an 
important cause of high-frequency fatigue, and the Na+,K+-pumps are essential for the 
maintenance of contractile force in the physiological range of [K+]o” (Clausen et al. 
2007). While this indicates a strong connection between potassium and muscle fatigue, 
it is important to note that the correlation has been found between potassium and high-
frequency fatigue, such as maximal contraction. While intramuscular potassium 
deficiency may play an essential role in fatigue at maximal contraction, it may play a 
lessened role in fatigue during extended steady state exercise.  
While ATP provides energy for contracting muscle and has been examined as 
part of substrate level fatigue, its byproduct ADP plays an important role in metabolic 
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fatigue. ADP is known to work with ATP as a natural check and balance system, where 
excessive ATP encourages ATP hydrolysis and excessive ADP prevents critical steps in 
glycolysis and encourages ATP synthesis. This is directly tied to the idea of inorganic 
phosphate, as ATP hydrolysis results in ADP and Pi. According to McLester, “Pi 
release is coupled to the powerstroke of the crossbridge cycle. The accumulation of Pi 
during exercise would lead to a reversal of its release step, therefore causing a 
decrement in force production capability” (McLester 1997). He goes on to conclude that 
“Pi accumulation is probably the largest contributor to the fatigue process in exercise of 
any duration” (McLester 1997). He continues on to detail that ADP plays a role not only 
in a “reduced oscillatory power output”, but in a “slowing of the rate constants (and 
therefore a decrease in the maximal velocity of shortening” (McLester 1997). This 
experiment suggests that at any intensity or duration, both ADP and Pi play an essential 
role in causing muscle fatigue. If there is a convenient method of measuring either of 
these metabolites in an accurate and non-invasive manner, they could potentially play 
an important role in predicting muscle fatigue during steady state exercise.  
It is important to note that external factors may play a large role in influencing 
fatigue. In high altitude environments, muscles may not have access to the amount of 
oxygen necessary for normal aerobic respiration, which may lead to fatigue (Hall 2013). 
Dehydration can be a major cause of fatigue, as hydrolysis is an extremely common 
reaction involved in metabolism (Hall 2013). In addition, dehydration may lead to poor 
blood flow and impaired cognitive function, making exercise more difficult. Finally, 
heat may play a major role in fatigue, as it may influence dehydration or reaction speeds 
(Hall 2013). While these three factors may play a significant role in muscle fatigue, 
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dehydration and altitude should be controlled for in any experiment. For this reason, 
heat production seems to be the most influential and potentially useful external cause of 
fatigue during steady-state exercise within a lab setting. A relationship between muscle 
heat production and muscle fatigue is an area that should be explored further.  
Finally, lactate has often been thought to be a cause of muscle fatigue. As 
discussed in the section on glycolysis, the function of lactate in the body is as a 
byproduct of anaerobic respiration. Theoretically, when an extended steady-state 
protocol is at too high of a workload for a subject, they will need to use both aerobic 
and anaerobic respiration to produce the necessary amount of ATP. This will lead to a 
buildup in lactate in the muscle. This lactate has long been thought to have an inhibitory 
effect on muscle contraction. However, Westerblad et al. note that “the temporal 
connection between impaired contractile function during fatigue and reduced pH is not 
always present” (Westerblad et al. 2002), noting however that “an alternative 
mechanism by which lactic acid formation may impose a limit on performance is during 
long-lasting types of exercise in which glycogen depletion is a key factor” (Westerblad 
et al. 2002), in which lactate causes an increased rate of glycogen store depletion 
because “the total amount of ATP produced from the stored glycogen is lower than with 
complete aerobic breakdown” (Westerblad et al. 2002). The connection between lactate 
threshold, acidosis, and muscle fatigue is explored more in depth later as well. 
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The role of pH 
Intro to pH 
The pH scale is a measurement of the concentration of hydrogen ions in a 
solution. It is represented by a logarithmic scale from one (acidic) to fourteen (basic), 
with seven being neutral. Chemical reactions are often heavily impacted by the pH in 
which they take place. One clear example is with hydrolysis reactions, as hydrogen ions 
produced through hydrolysis can build up and increase the energy requirement for 
further reactions to occur. The human body has a wide range of pH conditions, and each 
is optimized for its specific location. Digestive enzymes are optimized for the acidic 
conditions they are found in, while other reactions such the synthesis of ATP in liver 
mitochondria have multiple optimum pH values, corresponding to the different enzymes 
present (Myers and Slater 1957). Specifically in skeletal muscles, there are a few main 
factors which influence intramuscular pH. One of the largest factors has been thought to 
be lactic acid, which was thought to be produced during anaerobic respiration and 
dissociate into lactate and a hydrogen ion. However, modern research has discredited 
this idea, as lactate is produced, not lactic acid. The acidifying effects of lactate are still 
under debate. Potassium ions, creatine phosphate synthesis, and the bicarbonate buffer 
system also impact muscular pH. Blood pH tends to stay around 7.35 during rest, but 
may change during exercise as metabolites are produced and moved into the blood 
(Soller et al. 2007). It is important to note that while blood pH may often be a good 
predictor of intramuscular pH, it has been shown that exercise under certain 
circumstances can lead to significant differences between venous and intramuscular pH 
(Soller et al. 2007).  
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Old Ideas on pH 
In the past, it has often been standard practice to teach that pH is at least 
partially the cause of muscular fatigue. There were a number of theories on mechanisms 
through which this could occur, including troponin C becoming less available for 
calcium binding, chloride ion permeability of muscle membrane decreasing, 
interruption of ATP hydrolysis during cross bridge cycling, or lessening the amount of 
calcium released from the sarcoplasmic reticulum.  
One of the difficulties in such research was finding a practical method of 
studying pH in muscle during exercise. In 1939, Dubuisson helped to pioneer a method 
for measuring pH on the surface of skeletal muscle using an electrometer and a 
galvanometer. In this study, he recognized that past use of this technology had been 
“recording not only the changes in pH, but also variations in tissue polarization” 
(Dubuisson et al. 1939). Dubuisson claimed to have improved on these methods, such 
that there was “an excellent correlation” between “this recording technique [and] the 
results of chemical analyses made immediately after the experiment on the same 
muscles” (Dubuisson et al. 1939). While previous methods had only allowed pH 
measurement by chemical analysis at a single time point, Dubuisson helped to pioneer 
the idea of measuring pH throughout the duration of exercise.  
During the period of time from the 1930s through the 1970s, in vitro animal 
muscle was a major focus of muscular fatigue research. Following in the steps of 
Dubuisson, Disteche in 1960 went on to examine pH during both muscle twitch and 
tetany.  In this study, tortoise muscle at 22C was examined during “single isometric 
twitches” (Disteche 1960). Using the technology of Dubuisson, Disteche claimed that 
 19 
 
by “knowing the cellular carbon dioxide/bicarbonate ratio, the pK of carbonic acid, and 
the retention factor which accounts for other buffer systems”, one would be able to 
solve for the amount inorganic phosphate split from ATP by calculating the amount of 
hydrolysis of phosphate bonds from the remaining pH change in the muscle(Disteche 
1960).  In this experiment, Disteche found that during tetany the muscles he was 
examining reached a steady state, where he claimed the “absorption of H+ 
overcompensates the H+ liberation after a few stimuli” (Disteche 1960). Disteche ended 
up concluding that there was a “good qualitative agreement between the H+ production 
and the heat production during tetanus” (Disteche 1960). Overall, this experiment was 
useful for improving the method of measuring pH in muscle during exercise, and for 
demonstrating the relationship between muscular activation and pH.  
In June of 1967, Carter et al. improved further on this technique by using 
multiple-barreled electrodes. In the past, intracellular pH had been measured through 
indirect techniques, such as the Disteche study above where much had to be controlled 
for in order to estimate actual pH. According to their study, they used single barreled 
electrodes for the “determination of resting potential and intracellular pH with a 
minimum of cellular injury”, double barreled electrodes which by use of a reference 
were able to measure intracellular pH independent of transmembrane potential, and 
triple barreled electrodes which allowed for measurement during “controlled 
hyperpolarization or depolarization of the cell membrane” (Carter et al. 1967). During 
this study, Carter et al. found that they could only replicate pH values in previous 
experiments with “inadequately insulated electrodes” (Carter et al. 1967). They went on 
to examine rat thigh muscles in vivo while the exposed muscle was “continuously 
 20 
 
perfused with castor oil” at 37C (Carter et al. 1967). They ended up concluding that 
“H+ of intracellular and extracellular fluid was in electrochemical equilibrium at all 
levels of [transmembrane potential]” which they claimed implied that “the determinants 
of intracellular pH are the transmembrane potential and the blood pH” (Carter et al. 
1967). This study not only discounted some results from past studies, but made the 
assertion that there were large external factors influencing intracellular pH in skeletal 
muscle. 
In 1967, Hutter and Warner examined chloride conductance in frog Sartorius 
muscle using a similar micro-electrode technique. They noted that while skeletal 
membrane potential had once seemed insensitive to outside chloride ion concentration, 
research in the 1950s had shown that membrane potential was largely influenced by 
chloride concentrations at certain pH levels (Hutter and Warner 1967). By artificially 
controlling the pH of the solution that the frog muscle was kept in, they attempted to 
show a correlation between acidic conditions and muscle fatigue. According to their 
data, they found that alkaline conditions improved chloride conductance, and vice versa 
(Hutter and Warner 1967). They went on to conclude that “even moderate extracellular 
accumulation of hydrogen ions could produce an appreciable reduction in chloride 
permeability”, continuing on to suggest that in situations of muscle fiber depolarization 
and swelling, “a simultaneous fall in pH would produce a useful retardation of only 
slowly reversible osmotic changes” (Hutter and Warner 1967). Because chloride ions 
are closely tied to membrane excitation in skeletal muscle, decreased permeability of 
these ions in acidic conditions could theoretically lead to muscular fatigue. 
 21 
 
In a 1980 study by Stevens, frog Sartorius muscles were isolated and exposed to 
baths of either pH 7 or pH 8 at 22°C (Stevens 1980). The muscles were then electrically 
stimulated, and force production was measured. Stevens found that “presoaking in pH 8 
saline increased time to 50% fatigue by almost 50% in experiment A and 35% in 
experiment B” (Stevens 1980). This led to the conclusion that “the present experiments 
demonstrate that one important factor [in fatigue] is the pH of the external 
environment” (Stevens 1980). This study agreed with many others at the time, which as 
a combined body of evidence seemed to strongly support the idea that pH played a 
major role in muscle fatigue.  
However, research contradictory to these ideas also arose during this time, such 
as Kindermann et al. in 1977. In this experiment, a bicarbonate and Tris-buffer 
combination was given to males during a 400m run (Kindermann et al. 1977). The 
results suggested that “run time, maximal lactate concentration and heart rate remained 
unchanged after the buffer infusions” (Kindermann et al. 1977). However, the change in 
pH was only measured to be 0.1 after the infusion, and only 10 subjects were used. Still, 
Kindermann et al. concluded that “the importance of pH as the performance limiting 
factor must be questioned” (Kindermann et al. 1977). While this study was far from 
disproving the role pH played in muscular fatigue, it suggested that the results of past 
studies on pH may not translate to in vivo implications. 
More recently, these past studies have been criticized for a number of reasons. 
Very few studies took place in human models, and very few took place in vivo. Many of 
the studies used extreme pH values that could never be obtained within the body to get 
their results. In order to keep animal muscles stable in vitro, it was necessary to perform 
 22 
 
many of these experiments at temperatures far below physiological levels. Stackhouse 
et al. bring up a number of studies which show that “when muscle is studied at 
temperatures that are close to the normal body temperatures of living organisms, the 
effect of a decreasing pH on maximum isometric tension and shortening speed is greatly 
reduced” (Stackhouse et al. 2001). 
Modern research on pH causing fatigue 
While the causes of muscular fatigue are still up for debate, research in the past 
twenty years has largely shifted to the viewpoint that pH does not play a major direct 
role in causing muscular fatigue. Improvements in technology and methodology have 
led to more research in vivo, and many results from past studies have been discounted 
due to flawed methodology. Still, it is accepted that acidosis does have some 
physiological impacts in muscle. As Allen et al. stated in 1995, pH “reduces maximal 
Ca(2+)-activated force and Ca2+ sensitivity, slows the maximal shortening velocity and 
prolongs relaxation. However, acidosis is not the only metabolic change in fatigue 
which causes each of the above” (Allen et al. 1995). While these functions are accepted, 
there is contradictory research on the extent of these impacts as far as influencing 
fatigue.  
A review of literature on pH and muscle fatigue reveals that a significant 
number of sources stating the fatigue inducing effects of muscular acidosis are 
physiology textbooks. It is difficult to find an article on pH and fatigue that does not 
begin with some variation of ‘Intracellular acidosis has long been thought to be a cause 
of muscle fatigue’, many citing a range of textbooks over the past 60 years (Stackhouse 
et al. 2001). Peer reviewed articles strongly supporting the role of acidosis in muscular 
 23 
 
fatigue tend to be outdated, while more modern research on pH influence on fatigue 
takes a more cautious approach to the extent of the effects. Still, there is a large body of 
modern research which suggests that pH has at least some inhibitory effect on muscular 
performance. 
In a 2012 review of biomarkers to use for the determination of muscular fatigue, 
multiple different biomarkers are discussed, including lactate and pH. This study first 
mentions the fatigue inducing effects of acidosis by stating “multiple mechanisms of 
fatigue, of which the most important include: 1. Acidosis and depletion of ATP…” 
before continuing on to other biomarkers unrelated to pH (Finsterer 2012). This paper 
makes the important distinction that the exact causes of muscle fatigue are currently 
unknown, and that it is necessary to look at multiple possibilities. However, it supports 
the idea that acidosis causes fatigue by stating “even minimal decrease in muscle pH 
interferes with cross-bridge binding and ATPase activity due to competitive binding and 
reduced enzyme function” (Finsterer 2012). It continues on to state that “decreased 
intracellular pH may additionally impair oxidative enzyme activity and may adversely 
affect ryanodine receptor function” (Finsterer 2012). It is clear that even in 2012, the 
idea that pH causes muscle fatigue is both prominent and has not been categorically 
disproved. This paper further enforces the correlation between lactate threshold and 
muscle fatigue by stating “lactate appears to be a promising biomarker of muscle 
fatigue if workload conditions are standardized” (Finsterer 2012). 
 In a 2004 study by Robergs et al., the argument is made that there is no evidence 
for lactic acidosis, or any link between the supposed lactic acidosis and any metabolic 
acidosis (Robergs et al. 2004). There is a systematic review of the biochemistry behind 
 24 
 
lactate production which will be discussed later upon examining the temporal link 
between lactate threshold, acidosis, and muscle fatigue. The work even goes on to state 
that lactate production retards metabolic acidosis (Robergs et al. 2004). However, the 
part of this study which is relevant to this section is where the authors conclude that “if 
muscle did not produce lactate, acidosis and muscle fatigue would occur more quickly 
and exercise performance would be severely impaired” (Robergs et al. 2004). Whether 
or not the statement is true, there is a clear implication that acidosis causes muscular 
fatigue.  
 While many of the more recent studies which insinuate pH plays a role in 
causing muscular fatigue are based on past research, there is little novel data supporting 
that connection. Instead, the focus of a majority of current research on the topic either 
supports the idea that pH does not play a significant role in causing muscular fatigue, or 
the idea that muscular acidosis actually helps to prevent muscular fatigue. A 2014 study 
by Siegler et al. specifically examines “the effect of pH on fatigue” on human muscle in 
vivo (Siegler et al. 2015). Eight males performed three trials of submaximal isometric 
contractions, under a control condition as well as acidosis induced by ingested ammonia 
chloride and alkalosis induced by ingested sodium bicarbonate (Siegler et al. 2015). 
While muscular pH was not actively measured during the exercise protocol, Siegler et 
al. worked off of past research which suggested that blood pH would have an impact on 
both “central and peripheral factors associated with fatigue and force production” 
(Siegler et al. 2015). This study found that “calf fatigue associated with intermittent, 
isometric contractions to task failure is unaffected by alterations in pH” (Siegler et al. 
2015). There is a serious limitation when applying results of systemic acidosis induced 
 25 
 
by ingestion to the function of acidosis within exercising muscle. However, two 
strengths of this study are that it occurred both in vivo and in a human subject. This 
heavily suggests that while acidosis may have a multitude of intramuscular effects, the 
combined effect may well be neutral. 
 In a 2004 study by Pedersen et al., intracellular acidification is acknowledged as 
a commonly thought cause of muscle fatigue. Pedersen then goes on to detail an 
experiment which implies that acidosis protects against muscle fatigue. In this 
experiment, rat extensor digitorum longus was prepared such that the muscle could be 
stimulated at any individual step of the excitation-contraction coupling process 
(Pedersen et al. 2004). By doing this, Pedersen et al. were able to demonstrate that 
“force responses to [action potentials] in the T system elicited by electrical stimulation 
did display pH dependence” (Pedersen et al. 2004). This data suggested that 
“intracellular acidosis protects against the loss of force caused by depolarization” which 
may be due to “enhanced excitability of the T system” (Pedersen et al. 2004). In order 
to elucidate this mechanism, Pedersen et al. performed an additional experiment in 
which superphysiological levels of Cl- were introduced to the rat muscle in order to 
enhance the effects of chloride ions on depolarization of the T system. This setup 
resulted in significantly larger drops in force in alkaline than acidic conditions (7.1 and 
6.6 respectively) (Pedersen et al. 2004).  They concluded by stating that “in the 
presence of Cl-, intracellular acidosis increases the excitability of the T system in 
depolarized muscles[sic] fibers, thus counteracting fatigue at a critical step in ECC” 
(Pedersen et al. 2004). This experiment suggests that muscular acidosis helps prevent 
muscle fatigue by decreasing Cl- permeability. 
 26 
 
In a 2007 study, Lindinger cites previous research both by Sjogaard and within 
their own lab that suggests that fatigue is due to decreased sarcolemmal excitability. 
They state that fatigue is likely a mechanism to prevent damage that would occur to 
muscle cells if contraction continued. This paper reviews past literature to find that 
“acidosis counteracted the effects of increased extracellular potassium” on the 
sarcolemma in rat muscle (Lindinger 2007).  Lindinger went on to come to the 
conclusion that “increased extracellular acidity…similar to that seen during high 
intensity exercise” was able to combine with the effects of adrenaline to “stabilize 
membrane excitability” (Lindinger 2007), supporting the idea that “extracellular lactate 
accumulation has a protective effect on muscle excitability” (Lindinger 2007). They 
finished by acknowledging limitations, such as non-physiological temperatures and a 
limitation of the full range of physiological mechanisms due to in vitro research 
(Lindinger 2007). This experiment suggests that muscular acidosis helps to prevent 
muscular fatigue by decreasing the fatigue-causing effects of potassium ions. 
It is clear that there is still debate on the exact intramuscular effects of acidosis 
in relation to muscle fatigue. Current literature seems to be moving towards the position 
that pH does not play a major role in causing muscle fatigue. However, in our lab we 
are striving to anticipate muscle fatigue in exercising subjects, not to understand its 
cause. For this reason, it may be more pertinent to examine the temporal connection 
between acidosis, lactate threshold, and muscular fatigue. Even if acidosis were to have 
a preventative effect on muscular fatigue, if there were a significant correlation between 
when acidosis occurs and the onset of muscle fatigue, we would be able to use that 
connection to help prevent muscle fatigue in exercising subjects. 
 27 
 
Temporal connection 
 Before tackling the issue of a temporal connection between acidosis and fatigue, 
it seems relevant to discuss the correlation between lactate threshold and acidosis. As 
previously discussed, lactate threshold is closely related to anaerobic threshold. From a 
physiological standpoint, switching from only aerobic respiration to aerobic and 
anaerobic respiration is likely to mark the onset of muscle fatigue, or at least a point 
very close to it if there were a period of time where anaerobic respiration could be 
buffered. If this were true, then using pH to determine when a subject crossed their 
lactate threshold would allow a simple method of preventing subjects from experiencing 
muscle fatigue; as soon as a subject demonstrated that they had crossed their lactate 
threshold by a corresponding drop in pH, exercise intensity could be decreased to 
prevent fatigue and subject dropout. For this reason, it is relevant to discuss acidosis and 
fatigue in the context of lactate threshold, and to analyze if such a connection exists. 
Both sides of the argument on whether lactate causes or prevents acidosis agree 
on a couple of physiological concepts. First is the idea that anaerobic respiration does 
not produce lactic acid, but lactate. Second is the idea that muscles play not only an 
important role in lactate production, but also in lactate clearance. However, the 
disagreement comes in the form of both the number of hydrogen ions produced during 
lactate production and the physiological function of the lactate molecule itself. We 
previously discussed a 2004 study by Robergs et al. which made the claim that lactate 
production actually had a retardant effect on acidosis in muscles (Robergs et al. 2004). 
Robergs et al. supported this claim by analyzing the biochemical processes behind 
lactate production. They claimed that every two lactate molecules produced released a 
 28 
 
single hydrogen ion (Robergs et al. 2004). They went on to claim that “research also 
clearly discredits the interpretation of acidosis as being caused by lactate production”, 
saying that it was instead largely due to “nonmitochondrial ATP turnover” (Robergs et 
al. 2004). If true, this study would prevent the use of pH measurement as a determinant 
of lactate threshold.  
 In a 2005 paper, Lindinger et al. took direct issue with the Robergs paper and set 
out to disprove its assertions, stating Robergs’ argument “hinders interpretation and 
detracts from the demonstration of lactate- production independent of increasing 
intracellular [H+]” (Lindinger et al. 2005). They went on to argue that the Robergs 
paper violated both “conservation of mass and maintenance of electroneutrality in 
solutions” (Lindinger et al. 2005). The argument essentially hinges around the fact that 
lactate “is a strong acid anion that fundamentally alters the behavior of water” 
(Lindinger et al. 2005). Because of this, they argue that “the accumulation of lactate- 
within skeletal muscle directly contributes to intracellular acidosis” (Lindinger et al. 
2005). If true, this paper would suggest that we can indeed assume intracellular acidosis 
is related to lactate production, and therefore lactate threshold.  
When presented with conflicting evidence, it is often useful to look at the effects 
of believing the incorrect interpretation in order to determine the proper course of 
action. If we incorrectly assume that lactate threshold and acidosis are correlated, we 
risk misattributing an onset of acidosis to lactate threshold and muscular fatigue, or 
missing the point at which the subject reaches their lactate threshold and begins 
experiencing fatigue. If we incorrectly assume that there is no correlation between 
acidosis and lactate threshold, we are then unable to state that sudden acidosis indicates 
 29 
 
both lactate threshold and the onset of muscular fatigue. While literature seems to 
indicate a correlation between acidosis and lactate threshold, I believe it is safest to 
operate under the assumption of no correlation and instead examine the temporal 
connection between acidosis and muscular fatigue independent of lactate threshold. If 
such a useful connection exists, whether the acidosis is caused by lactate threshold or by 
another cause becomes irrelevant.  
 There is a 2001 paper by Stackhouse et al. by the title of “challenging the role of 
pH in skeletal muscle fatigue” which provides an excellent introduction into the topic of 
pH, suggested mechanisms by which acidosis could cause fatigue and research which 
refutes each one, as well as a section titled “lack of temporal association” (Stackhouse 
et al. 2001). In this section, the following three studies are brought up as evidence, as 
well as a fourth which is not free for public access.  
The definition of a lack of temporal connection between acidosis and muscle 
fatigue used in the Stackhouse paper is borrowed from another paper by Saugen et al., 
who state “increases or decreases in metabolite levels [which] do not occur at the same 
time as increases or decreases in force-generating capacity” (Saugen et al. 1997). This 
definition limits temporal association to only existing in the case of an exact temporal 
correlation between acidosis and fatigue. In this 1997 study, eight subjects performed 
one-legged repetitive isometric knee extensions at 40% of maximum contraction for six 
seconds at a time until exhaustion. While this was happening, pH and other metabolites 
were measured every nine seconds. pH was measured using P-NMR, or Phosphorous-
31 nuclear magnetic resonance. They noticed an immediate uptick in pH at the onset of 
exercise, which they hypothesized to be due to hydrolysis of phosphocreatine (Saugen 
 30 
 
et al. 1997). After that, there was a steady drop in pH as exercise continued. However, 
there was a lot of variability, as some subjects experienced rapid drops in pH, while 
others experienced much smaller, steadier declines, as can be seen in Figure 1 (Saugen 
et al. 1997). 
   
Figure 1: pH and time during one-legged repetitive isometric knee extensions 
While some subjects did not experience significant changes in pH, the group as 
a whole went from a resting pH of 7.13±.02 to a pH at 25% of exercise duration of 
7.00±.06, with an insignificant decrease in pH from that until exhaustion, as can be seen 
in Table 1 (Saugen et al. 1997). 
 
 31 
 
Table 1: pH decreases during exercise 
They noted in their data that “in some subjects RIE could be continued for 10-15 min. . 
.without further changes in pH or ATP” (Saugen et al. 1997).  In the discussion, they go 
on to suggest that the rest intervals between contractions “enable[d] sufficient aerobic 
ATP resynthesis, in keeping with previous results. . . showing a very moderate rise in 
muscle lactate” (Saugen et al. 1997). This research does not indicate any sort of useful 
temporal connection between pH and muscle fatigue. However, this may be due to 
exercise intensity being only at 40% of maximum effort, as there may be no transition 
into anaerobic respiration. Because this experimental protocol required 40% max effort 
and for the subject to maintain contraction for six seconds before resting for two, it may 
give significantly different results than dynamic knee extension with 65% max effort, 
no isotonic portion, and shorter rest periods between contractions.  
Wong et al. specifically looked at patients with chronic fatigue syndrome when 
determining a temporal relation between acidosis and fatigue. 22 CFS patients were 
compared to 21 healthy adults in a protocol of “dynamic, graded, plantar flexion” with a 
“constant repetition rate of 30 cycles/min against resistance that was increased at 2 
kg/min” until exhaustion (Wong et al. 1992). P-NMR was used to acquire a range of 
data, including pH. They went on to conclude from their data that “the degree of change 
in PCr, Pi, and pH from rest to peak dynamic exercise was quantitatively large, and 
equal, in both study groups” (Wong et al. 1992). While the focus of this study was to 
determine differences between healthy populations and CFS patients, there was 
evidence of a significant decrease in pH right before exhaustion when compared to rest. 
 32 
 
 Additional evidence against a temporal connection between acidosis and fatigue 
comes in a 1993 study by Degroot et al., in which five subjects performed an exercise 
protocol of “maximal isometric foot plantar flexion sustained for 4 minutes” (Degroot et 
al. 1993). P-NMR spectra were obtained in order to analyze an array of metabolites. 
Results showed that “[H+] decreased immediately after the onset of exercise, but then 
rose steadily until the end of exercise to a value of . . . pH6.47±.04” compared to a 
control value of pH 7.09, as can be seen in Figure 2 (Degroot et al. 1993).  
 
Figure 2: H+ concentration and time during maximal isometric foot plantar flexion  
Notably, there was a significant increase in pH during the first 20 seconds of 
exercise (likely due to PCr hydrolysis), with a concurrent steady decline in MVC. 
Overall, this study found that “although the decline of force and increase in [H+] may 
 33 
 
be associated later during exercise, during the initial 10 seconds force declines while 
[H+] decreases” (Degroot et al. 1993). 
 Returning to the Stackhouse paper from above, the three previous papers 
are used to make the assertion that “the results of these studies. . . demonstrate that in 
certain phases of fatiguing exercise, there is a clear lack of temporal association 
between changes in pH and changes in force” (Stackhouse et al. 2001). Using the -
definition given in the Saugen paper of a lack of temporal association being “increases 
or decreases in metabolite levels [which] do not occur at the same time as increases or 
decreases in force-generating capacity”(Saugen et al. 1997), it is clear that this is a 
reasonable conclusion; all three papers showed that changes in pH did not exactly align 
with changes in force production, especially at the onset of and recovery from exercise. 
However, all three papers showed a significant drop in pH between rest and peak 
exercise. In addition, all three studies were performed at exercise intensities other than 
steady state exercise. In fact, the Degroot paper specifically states that “one long-
standing hypothesis has been that fatigue occurs as a result of a rise in intracellular 
[H+], and this has been supported by various studies which have employed steady state 
or prolonged exercise”, before going on to explain why this experiment would be 
different than ones which utilized steady state protocols (Degroot et al. 1993).  With 
these papers, we can come to a couple important conclusions. Firstly, the time course of 
pH does not exactly follow muscle fatigue, especially at onset or recovery from 
exercise. Secondly, as examined above, acidosis is likely not the cause of muscle 
fatigue. However, there does seem to be a significant reduction in pH at peak exercise 
when compared to rest. Even if acidosis does not cause muscle fatigue and does not 
 34 
 
always occur at the exact same time as muscle fatigue, if there is a general correlation 
between acidosis and fatigue at any time-point during exercise, it may still be useful for 
preventing fatigue during a steady state protocol. 
The next step is then to examine the limited data on pH during steady state 
exercise in order to determine if there is a usable connection between pH and muscle 
fatigue. A study by Street et al. in 2001 conducted a study with a protocol much closer 
to steady-state conditions. Six subjects “performed one-legged knee extensor exercise” 
and “were required to maintain a cadence of 60 r.p.m for 5 min duration at each 
workload” for workloads of 30, 50, and 70W (Street et al. 2001). pH was studied using 
microdialysis throughout the exercise protocol. At rest, mean interstitial pH was 7.38. 
Street et al. found that “exercise induced a reduction in muscle interstitial pH in all six 
subjects and at all intensities”, as well as “a correlation between power output and peak 
acidification” in each subject (Street et al. 2001).  
 35 
 
 
Figure 3: Individual continuous pH response during one legged knee extensor exercise 
Figure 3 shows each subject’s individual pH response to exercise, where it is 
evident that pH decreases according to power output throughout the entire duration of 
exercise which does not end in exhaustion (Street et al. 2001). Figure 4 shows the 
relationship between power output and interstitial pH (Street et al. 2001).  
 36 
 
 
Figure 4: pH and power output during one legged knee extensor exercise 
The paper concludes by stating “the present study demonstrated that interstitial 
pH is continuously decreasing during muscle activity”, noting that “pH was correlated 
with power output” (Street et al. 2001). The main issues preventing this study from 
being directly applicable to our needs are the small sample size, non-relative workloads, 
and the fact that exercise was not performed until fatigue (except for one trial for one 
subject). Because of these limitations, we are unable to see if there are significant 
changes in pH right before exhaustion during steady state exercise. Still, this study 
provides evidence that pH is continually decreasing during steady state exercise, and 
suggests that pH measurement could be a useful tool for determining muscle fatigue, 
especially if these results are replicable within subjects. 
 Further evidence of the use of pH in steady state exercise is presented in a 1985 
paper by Wilson et al., in which “nine patients with chronic congestive heart failure” 
and eight controls were put through an exercise protocol involving steady state exercise 
 37 
 
of the forearms (Wilson et al. 1985). P-NMR was used to measure pH levels throughout 
the protocol, which consisted of “wrist flexion every 5 sec for 7 min” at 1, 2, and 3 J. 
They found that “exercise resulted in a decrease in pH only at 0.6W”, or only at the 
highest workrate. This can be seen in Figure 5 (where the dashed line represents CHF 
patients and the solid represents control), where pH remains fairly even between rest 
and the lower workrates in healthy patients, but then drops significantly for the highest 
workrate (Wilson et al. 1985). In the lower half of Figure 5, fatigue is ranked on a 
subjective perceived scale by the subject, with 0 being no fatigue and 4 being highest.  
 
Figure 5: pH, power output, and fatigue during steady state wrist flexion 
Fatigue increases with workload as expected, and correlates nicely with the drop 
in pH after exercise (Wilson et al. 1985). Due to the use of P-NMR, this study does not 
 38 
 
have continuous measurement of pH throughout the exercise protocol. However, it does 
suggest that there may be a steady-state workload which leads to a significant drop in 
pH relative to lesser workloads. This is very promising for future research on the 
connection between a measurable sudden acidosis event and the onset of fatigue.  
 One of the most promising studies occurred in 1988, by Miller et al. In this 
study, the exercise protocol consisted of both a 4 minute sustained maximum voluntary 
contraction and an intermittent protocol, of which the latter is more relevant to our work 
(Miller et al. 1988). The intermittent protocol consisted or contracting and relaxing the 
adductor pollicis once every 10 seconds for 5 minutes at 75% of their MVC. This was 
repeated eleven times without rest periods, with each trial occurring at a different 
contraction/relaxation split (6s contraction and 4s rest, 3s contraction 7s rest, etc.) 
(Miller et al. 1988). Miller et al. state that “analysis of 1-min spectral blocks indicated 
that a steady state was almost always reached after 1 min” (Miller et al. 1988). After 
analyzing the data, the conclusion was that “during intermittent exercise, pHi gradually 
dropped to 6.55±.03 [compared to a resting pH of 7.08±.04] and then gradually returned 
to control values by 40 min” (Miller et al. 1988). In Figure 6 where triangles represent 
pH and squares represent MVC, it is evident that during the steady state protocol, pH 
and MVC displayed qualitatively similar changes throughout the protocol (Miller et al. 
1988). Specifically, a significant drop can be seen in both from rest to the onset of 
exercise. From there, pH steadily drops along with MVC until the cessation of exercise, 
at which point pH begins to recover and MVC quickly follows (Miller et al. 1988). The 
lag that MVC experiences in recovery supports the lack of exact temporal association, 
but the overall qualitative connection is clear. 
 39 
 
 
Figure 6: pH over time during steady state adductor pollicis contraction 
This correlation is even more evident in Figure 7 (Miller et al. 1988). 
 
Figure 7: Maximal volumetric contraction and hydrogen concentration during steady 
state adductor pollicis contraction. 
 A regression analysis was performed between the two variables, and an r value 
of 0.77 was found, indicating a strong linear relationship between MVC and pH (Miller 
 40 
 
et al. 1988). This study indicates that while acidosis may not cause fatigue or even be 
directly temporally associated with it, there seems to be a strong linear relationship 
between acidosis and fatigue during steady state exercise protocols. This is extremely 
encouraging for future research, as it makes clear that measurement of pH may well 
lead to useful extrapolations to fatigue. 
 These three papers examining acidosis during steady state exercise provide a 
significant body of evidence suggesting that pH measurement may be a useful tool 
during this type of exercise. While there are a lot of unknowns, it seems clear that for a 
portion of steady state exercise, pH most likely decreases at a steady rate. Further, it 
appears evident that higher workloads lead to a more significant decrease in pH than 
lower workloads. Finally, although causation and temporal association have not been 
shown, it seems as though pH is closely related to muscle fatigue as determined by 
continuous measurement of maximal volumetric contraction. Assuming the technology 
is practical and available, it seems to be worthwhile to more closely examine continuous 
pH response to steady state exercise at levels which may induce fatigue.  
 41 
 
Methods of measuring pH 
There are a couple goals we must have when determining the best technology 
and methodology to measure pH during steady state exercise. First is the potential for 
nearly continuous measurement; more frequent measurements allow for a greater 
chance of sensing significant changes in pH at any given time point. The second is 
minimal invasiveness in order to both allow for use during most experiments and to 
improve subject experience. The third is demonstrated, repeatable accuracy in 
measuring the intended value. The fourth is minimal equipment needed; technology that 
is prohibitively expensive or requires special staff to operate will not be practical to use 
during a standard exercise protocol.  
Two techniques used in the past to measure pH are venous blood samples and 
muscle biopsies. In the Street paper discussed previously, the argument is made that 
since venous blood is a mixture of metabolites from the working muscle and blood 
returning from other non-working muscle, there must be some difference between blood 
and interstitial pH (Street et al. 2001). It goes on to state that due to differences found 
between blood and interstitial lactate levels, “it may, therefore, also be expected that 
interstitial to venous pH gradients exist during exercise”, concluding this section with 
“it can be hypothesized that the exercise-induced changes in interstitial and venous 
blood pH are different and that the changes in venous blood pH underestimate the local 
interstitial pH changes” (Street et al. 2001). While venous blood draws are simple to 
perform, venous blood lacks accuracy in estimating interstitial pH and is impractical to 
take at regular short intervals throughout exercise. For this reason, venous blood draws 
are not practical for pH monitoring during steady state exercise. Similarly, muscle 
 42 
 
biopsies have been taken from exercising muscle and analyzed to determine pH levels. 
However, high accuracy in measuring interstitial pH is traded for not allowing 
continuous measurement, being highly invasive, and requiring special staff and 
equipment. For these reasons, muscle biopsy should not be considered a practical 
method of pH measurement during steady state exercise.  
A large number of the studies which occurred in the 20th century used glass 
microelectrodes to measure pH. As discussed in the history of pH measurement section 
earlier in this paper, this technique was used in human as well as other animal muscle, 
and has gone through a long period of technique refinement. There is a discussion of the 
method of using mircoelectrodes “for measuring potential or determining the free 
concentration of cytosolic constituents” in a paper titled “using microelectrodes” 
(Halliwell et al. 1987). While the methodology of using microelectrodes has existed for 
a century, nearly every subsequent paper does something to improve upon the errors of 
past studies. This paper documents the five main sources of error when using 
microelectrodes as follows: 
“(1) Varying tip potentials of the ME. (2) Varying junction potentials. 
(3) Asymmetry of electrode reference potentials and their dependence on 
salt concentration in the bath solution. (4) Inadequate amplifier 
frequency responses when monitoring fast signals. (5) Errors in potential 
measurement when injecting current because of ‘bridge balance’ or ME 
resistance change, or due to too high a switching rate when using the 
discontinuous current injection method” (Halliwell et al. 1987) 
The benefits of this technology are that the shortcomings are well understood 
and documented so that use of the equipment will likely give results that closely align 
with what is actually meant to be measured. Techniques such as the single/double/triple 
barrel microelectrode as discussed in the Carter paper in the section on pH causing 
 43 
 
fatigue exist which make the technology highly adaptable to specific needs (Carter et al. 
1967). The main issue is that study of intramuscular pH requires exposure of the 
exercising muscle for accurate measurement. Surface and skin membrane potential 
measurements have been taken, but relationships between those and intracellular 
measurements are far from conclusive. For this reason, this technique has not been used 
for exercising human muscle in the past, and is likely not practical for pH monitoring 
during steady state exercise.  
 A technique which improves upon the weaknesses of venous blood draw (non-
continuous, not an accurate measurement of intracellular pH) is microdialysis. With this 
method, after injection of a local anesthetic a microdialysis probe is introduced into the 
exercising muscle using an introducing needle, which is subsequently removed. A pH-
sensitive dye can be mixed with a saline solution and infused through the microdialysis 
probe. When used in conjunction with a spectrophotometer, constant monitoring of 
intracellular pH can occur. This method was used to great effect in the Street paper 
discussed earlier. This study improved upon past methods of microdialysis which 
actually showed alkalization during muscle activity by adding a bicarbonate buffer to 
the perfusate at physiological levels, or about 25 mM (Street et al. 2001). This method 
has the benefit of providing a constant stream of data for analysis, allowing for greater 
temporal accuracy than any other current method (Street et al. 2001). Microdialysis is 
somewhat invasive, but does not require highly specialized training or inhibit normal 
exercise. However, this method has not been used extensively in the past, so it is 
difficult to know if current techniques provide perfectly accurate results. In exercise 
protocols which do not require invasive equipment, introducing anesthesia and a probe 
 44 
 
inside the exercising muscle seems to be excessive for our purposes. However, the 
method does seem to be very effective and is worth consideration in certain situations. 
 Many of the more recent studies have utilized P-NMR, or phosphorous-31 
nuclear magnetic resonance.  In the Miller paper discussed previously, P-NMR was 
used specifically to measure human intramuscular pH during steady state exercise. 
According to Maryellen Nerz-Stormes of Bryn Mawr college, NMR “is based on the 
fact that when a population of magnetic nuclei is placed in an external magnetic field, 
the nuclei become aligned in a predictable and finite number of orientations. For 1H 
there are two orientations” (Nerz-Stormes 2009). If you picture a compass, there are 
two stable positions for the needle; with the south end facing north as natural (the alpha 
form) or with the north end facing so perfectly north that the forces pushing it to either 
side balance out (the beta form). By putting energy into the system, the nuclei are 
switched to the beta form, and upon returning to the alpha orientation create a 
measurable change in the magnetic field. This can be used to calculate H+ ion 
concentration assuming you can account for the other molecules present in the sample 
(Nerz-Stormes 2009). NMR using phosphorous is specifically used because it is easier 
to interpret than other methods of NMR. In the Miller study, P-NMR provided useful, 
significant data when examining intramuscular pH (Miller et al. 1988). However, P-
NMR requires around 30 seconds for each scan, during which time the muscle being 
examined can’t be moved. Additionally, P-NMR requires access to a superconducting 
magnet to generate the required magnetic field, such as an MRI machine. P-NMR has a 
lot of use in determining the role pH plays in the body and in the muscle. However, it is 
not a practical method of pH monitoring during steady state exercise.   
 45 
 
Near Infrared Spectroscopy is not a new technology. However, due to the 
complex nature of its functioning, new uses are still being discovered. Spectroscopy 
depends on chemical interactions within molecules. If a molecule is made of two atoms, 
the atoms are bonded to each other through an interaction of forces. These forces can be 
thought of as a spring between the atoms. This spring naturally stretches and relaxes, 
creating a vibration within the molecule. Adding more atoms creates a large number of 
unique ways in which the molecule can vibrate (Rupawalla et al. 2013). As with 
anything that can vibrate, there are specific resonant frequencies, in which the internal 
vibration of the molecule combines with external vibrations to cause a greatly 
magnified effect (Rupawalla et al. 2013). Near Infrared Spectroscopy uses 
electromagnetic waves near the infrared range of wavelengths. By projecting these 
waves onto a molecule and going through a range of wavelengths, it is possible to 
determine which wavelengths cause the molecule to resonate (Rupawalla et al. 2013). 
Because every molecule has different forces causing different internal vibrations, it is 
then possible to look at which wavelengths caused resonance, and reverse engineer 
what molecule is present (Rupawalla et al. 2013). 
 This technology was first utilized in the 1950s for chemistry applications. 
However, it has been recently found that the same technology could be used to measure 
pH levels in the muscle (Soller et al. 2008). Because pH of interstitial fluid is dependent 
on the relative concentrations of hydrogen ions and carbon dioxide, analyzing which 
molecules are present in the sample using the method described in the previous 
paragraph allows for analysis of interstitial pH (Soller et al. 2008). In a paper entitled 
“Noninvasive determination of exercise-induced hydrogen ion threshold through direct 
 46 
 
optical measurement”, Soller et al. used many of the concepts discussed above in order 
to determine if there is an H+ threshold during exercise which occurs at the same time 
as anaerobic threshold (Soller et al. 2008). Soller notes that “Near-infrared light passes 
through skin and subcutaneous fat and can be used to noninvasively measure metabolic 
parameters in muscle” (Soller et al. 2008). The experiment was designed around the 
hypothesis that “this methodology can be extended to determine a threshold based on 
the accumulation of hydrogen ions in the muscle interstitial fluid during graded 
exercise, the H+ threshold” (Soller et al. 2008). 
The exercise protocol consisted of both handgrip dynamometry with eight 
subjects and cycle ergometry with ten subjects. The handgrip protocol involved “four 5-
min bouts” of “2-s contractions with intervening 1 s of relaxation”, with workload 
determined by MVC (Soller et al. 2008). The cycle protocol involved “a graded 
exercise test. . . to maximal exertion” with 3 minute stages at 75 rpm and an increase of 
50W per stage (Soller et al. 2008). During both protocols, NIRS data was collected and 
blood samples were taken to determine lactate levels. As can be seen in figure 8, a very 
close relationship was found between NIRS H+ measurements and invasive controls, 
with an r2 value of 0.722 (Soller et al. 2008).  
 47 
 
 
Figure 8: Comparison between pH measured by NIRS and by traditional invasive 
methods 
In Figure 9, there are two things of note in the data of a single subject. First, 
there is a clear qualitative H+ threshold that occurs, with a sudden increase in H+ 
concentration at a certain exercise rate. Second, there is a clear qualitative connection 
between this H+ threshold and the lactate threshold (Soller et al. 2008).  
 48 
 
 
Figure 9: Proposed H+ threshold measured by NIRS in an exercising individual 
This data is only from a single subject and a general conclusion can’t be made, 
but the data is promising for future research. Soller et al. further found that H+ 
threshold and LT were highly correlated with an r2 value of 0.946 (Soller et al. 2008). 
They conclude by stating that “an accurate mathematical model was established relating 
pHm to near-infrared spectra of exercising muscle during handgrip dynamometry and 
that the resultant model can be used during graded cycle exercise to measure [H+]” 
(Soller et al. 2008). 
NIRS has been shown to provide fairly continuous measurement, be minimally 
invasive, accurately determine intramuscular pH, and do so without any prohibitive 
 49 
 
equipment or training. In addition, it has been used to determine the exact mechanism 
that we are looking for in our research, namely an H+ threshold that is related to both 
lactate threshold and possibly the onset of muscle fatigue. For these reasons, NIRS is 
the gold standard for monitoring pH during steady state exercise. The main drawback is 
that because this is a novel use of this technology, it is relatively unproven. If H+ 
thresholds are repeatable within subjects, we can theoretically use this technology to 
make sure that no subject crosses their lactate threshold, preventing subject dropout and 
confounding variables during aerobic steady-state exercise. 
Future directions for research 
 The Soller study discussed above is a very promising proof of concept, but 
additional research must be done. To determine a future direction for research, we have 
to ask what we know for sure, what we suspect, and what still needs to be tested. As I 
have systematically shown in this paper, there are a few facts that must be accepted 
until future research disproves them. First, evidence does not support the idea that 
acidosis is the primary cause of muscle fatigue. Second, evidence does not support an 
exact temporal connection between acidosis and lactate threshold. Third, evidence does 
not support an exact temporal connection between acidosis and muscle fatigue. 
However, it has also been shown that for the purposes of monitoring pH during steady 
state exercise to prevent muscle fatigue, an exact association is not needed, only a 
correlation between pH and muscle fatigue. We further know that for these purposes, 
past techniques of pH measurement like venous blood draws, muscle biopsies, and glass 
microelectrodes are impractical options. We have seen how P-NMR and microdialysis 
can both have limited use depending on the situation. We also know that preliminary 
 50 
 
data suggests that NIRS is the most promising way forward for pH monitoring during 
steady state exercise. 
 Due to a knowledge of physiology and past research, we suspect that at a certain 
steady-state exercise level, some physiological threshold is crossed which begins the 
process of muscle fatigue. We further suspect that this threshold is related to the switch 
between aerobic respiration to aerobic and anaerobic respiration. While not necessarily 
caused by or exactly in time with the anaerobic threshold, it would make sense if this 
threshold were at least somewhat correlated with the lactate threshold. Further, we 
suspect that around the time that this physiological threshold is crossed and muscle 
fatigue begins to set in, there may be a sudden build-up in H+ concentration, regardless 
of the cause and effect. Research by Soller et al. suggests that we can not only measure 
this build-up with NIRS, but according to our other suspicions we could then use this 
H+ threshold to prevent muscle fatigue in subjects. 
 There is much that still needs to be tested in the future based on these ideas. One 
of the first directions of research should be to determine if H+ thresholds are present 
during exercise protocols that we run in the lab, including dynamic knee extension and 
cycle ergometry protocols. This can be accomplished by running standard exercise 
protocols with a NIRS sensor attached, and looking for a sudden inflection in H+ 
concentration. If H+ thresholds are found within groups of subjects, the next step will 
be to determine if this data is useful for single subjects, by creating a method of 
determining when a subject has hit their H+ threshold. A steady state H+ concentration 
will need to be established in each subject, with a protocol to determine if a specific 
value lies above or below the H+ threshold. If these experiments are successful, the next 
 51 
 
step would be to find a correlation between H+ threshold and muscle fatigue. This could 
be accomplished with a cycle ergometry study, with one group of subjects exercising at 
a level just above their H+ threshold, and a control group exercising at a level just 
below their H+ threshold. If the experimental group fatigues significantly faster than the 
control group, it can then be concluded that NIRS is a useful tool to prevent subjects 
from experiencing fatigue during steady state exercise. If this is found, NIRS will prove 
to be an invaluable resource for not only our lab, but any lab involved in steady-state 
sub-anaerobic threshold exercise protocols. 
Conclusion  It has been systematically shown that the causes for muscle fatigue are still up 
for debate. However, evidence supports the idea that pH is neither causally nor exactly 
temporally associated with fatigue. However, evidence does suggest that pH 
measurement may still be useful for determining the onset of muscle fatigue during 
steady state exercise. There are a variety of methods that exist for measuring pH in the 
body, of which the most promising for our purposes are microdialysis, P-NMR, and 
NIRS. Of the three, NIRS appears to be the best choice for further research. Future 
research should focus on establishing the functionality of NIRS for determining the 
onset of muscle fatigue in individuals performing steady-state exercise protocols.   
  
52 
 
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