Received Date : 12-Jul-2016

Revised Date : 24-Oct-2016

Accepted Date : 28-Oct-2016
Article type : Review Article
Accepted Article
Biology of VO2max: looking under the physiology lamp

Carsten Lundby1, David Montero2 & Michael Joyner3.

1
Zürich Center for Integrative Human Physiology, Institute of Physiology, University of
Zürich, Switzerland; 2Department of Cardiology, University Hospital Zurich, Switzerland;
and 3Department of Anesthesiology, Mayo Clinic, Rochester, MN 55902, USA

Short title: Trainability of humans

Correspondence:

Prof. Carsten Lundby

University of Zurich
Institute of Physiology
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
Tel: +41 44 635 50 52
Email: carsten.lundby@uzh.ch

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/apha.12827
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 Abstract

In this review we argue that several key features of maximal oxygen uptake (VO2max) should
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underpin discussions about the biological and reductionist determinants of its inter-individual
variability: 1) Training induced increases in VO2max are largely facilitated by expansion of
red blood cell volume and an associated improvement in stroke volume, which also adapts
independent of changes in red blood cell volume. These general concepts are also informed
by cross sectional studies in athletes that have very high values for VO2max. Therefore, 2)
variations in VO2max improvements with exercise training are also likely related to
variations in these physiological determinants. 3) All previously untrained individuals will
respond to endurance exercise training in terms of improvements in VO2max provided the
stimulus exceeds a certain volume and/or intensity. Thus genetic analysis and/or reductionist
studies performed to understand or predict such variations might focus specifically on DNA
variants or other molecular phenomena of relevance to these physiological pathways.

Key words: Exercise, genetics, mitochondria, non-responders, performance, red blood cell
volume.

Introduction: Physiology, Reductionism & Street Lamps

Maximal oxygen uptake (VO2max) and its closely related clinical correlate cardiorespiratory
fitness are key determinants of both elite performance in endurance sports and mortality in
the general population (Pedersen and Saltin, 2015). In this review we argue the dominant and
deterministic physiological pathways that account for a vast majority of inter-individual
variability in VO2max are well known and center on total body hemoglobin content and peak
cardiac stroke volume and as a result cardiac output. In this context, searches for reductionist
explanations and more basic biological factors that might contribute to inter-individual
variability in VO2max and its response to training should focus on these physiological
pathways. We adapt the “street lamp parable” to highlight our position. In the street lamp
parable an intoxicated individual is searching for a set of lost keys under the street lamp
because that is where “the light is”. In most cases the street lamp parable is a cautionary tale
about observational bias. By contrast, in the case of VO2max failure to focus where the
physiological light has led to limited insight about any DNA variants and other molecular

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 mechanisms the might contribute to this critical physiological phenotype. Additionally, we
hope to use this physiological “light” to discuss two types of studies that seek to understand
the basic biology underpinning VO2max. One type is cross sectional studies including elite
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athletes who have decidedly extreme phenotypes due to whatever biological endowment they
possess plus years of prolonged and intense training. The other type of study is the classic
short term intervention in “average” citizens that seeks to understand the phenotypic changes
associated with periods of exercise training lasting a few months.

Red cell mass, total body hemoglobin, blood volume and stroke volume explain inter-
individual differences in VO2max

Figure 1 illustrates the clear relationship between VO2max expressed in l.min-1 and cardiac
output (l) and red blood cell mass (ml). Additionally acute interventional maneuvers that alter
these variables also typically alter VO2max in a way (Calbet et al., 2006b) consistent with
these cross sectional data. The key points of Figure 1, and we start our review with them, are
that the physiological pathways that dominate the determinants of VO2max are well known
and in a statistical context explain a very high fraction of the population variance. The clarity
of these physiological relationships is in stark contrast to studies on larger cohorts of elite
endurance athletes that show no clear genetic explanation for these findings (Rankinen et al.,
2016). By contrast Finnish cross country skier Eero Antero Mäntyranta (Winter Olympics
1960–1972; winning seven medals at three of them) had a variant in the erythropoietin
receptor that enhanced red blood cell production resulting in very high total body hemoglobin
and VO2max which together with high training volume and talent for the sport have
contributed to his astonishing achievements. In this context, it seems reasonable to propose
that any reductionist search for factors that explain inter-individual variability in VO2max and
how it responds to training or other conditions is likely to interact with these critical
physiological pathways.

Physiological adaptations of importance for improving VO2max

Regular exercise, particularly endurance training (ET), is associated with manifold

phenotypic modifications potentially influencing VO2max (Hawley et al., 2014, Hellsten and
Nyberg, 2015). These are conventionally systematized into two broader categories according

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 to their primary role in the oxygen (O2) transport and utilization chain: enhancing convective
O2 delivery or O2 extraction. The former mainly comprise increases in blood volume (BV),
O2-carrying capacity of the blood and cardiac output; the latter generally includes skeletal
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muscle adaptations such as augmented capillarization and mitochondrial content (Kjellberg et
al., 1949a, Hoppeler and Weibel, 1998). Adaptations related to muscle motor recruitment
were theorized but recently refuted to govern VO2max (Hawkins et al., 2007, Brink-Elfegoun
et al., 2007), thus they are not covered in this review, which as a matter of fact aligns with
and expands upon the ‘classical’ view of VO2max as being primarily limited by the circulatory
capacity to deliver O2 to working muscle (Hill and Lupton, 1923, Bassett and Howley, 1997,
Levine, 2008). The reader is referred to a comprehensive review by Levine (Levine, 2008)
and recent debates discussing theories and facts for VO2max limitation (Wagner, 2015,
Lundby and Montero, 2015).

Dissecting the relative roles of training adaptations in determining VO2max improvements is a

fundamental topic in exercise physiology with tangible impact on exercise prescription
targeting cardiorespiratory fitness (Mezzani and Guazzi, 2016). What follows hereunder is an
integration of studies assessing the effects of ET on potential determinants of and their impact
upon VO2max in untrained but otherwise healthy humans. Nearly half a century ago, the
question arose whether the increase in VO2max following ET is underlain by adaptations
characterized by ‘central’ and/or ‘peripheral’ nature, respectively and primarily reflected by
the 2 components of the Fick equation (Figure 2): maximal cardiac output (Qmax) and
arteriovenous O2 difference (a-vO2diff) (Ekblom et al., 1968, Saltin et al., 1968). These early
studies reported an increase in Qmax alone or along with a-vO2diff following 8-16 weeks of ET
in few (< 10) healthy young individuals (Ekblom et al., 1968, Saltin et al., 1968). Subsequent
studies also had small sample sizes but confirmed prevalent increases in Qmax with variable a-
vO2diff in response to diverse ET interventions ranging from 5 to 52 weeks of duration, in
mainly untrained individuals accross all ages (Klausen et al., 1982, Spina et al., 1992, Beere
et al., 1999b, Helgerud et al., 2007, Murias et al., 2010b, Weng et al., 2013, Bonne et al.,
2014, Jacobs et al., 2013, Macpherson et al., 2011, Wang et al., 2014b, Ehsani et al., 1991,
Fujimoto et al., 2010, Haennel et al., 1989, Hijazi et al., 1998, Marshall et al., 2001, Morris et
al., 2002, Murias et al., 2010a, Spina et al., 1993a, Spina et al., 1993b, Spina et al., 1998,
Wang et al., 2014a). Pooled evidence suggests that with training interventions lasting ≥ 12
weeks the effects on a-vO2diff become evident (Montero et al., 2015d, Montero and Diaz-
Canestro, 2015). Importantly, these meta-analyses reveal a linear relationship between

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 VO2max and Qmax but not a-vO2diff increases (Montero et al., 2015d, Montero and Diaz-
Canestro, 2015). This highlights the predominant dependence of any improvement in VO2max
induced by ET on Qmax, concurring with the classic view of VO2max being basically
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determined by the capacity of the cardiovascular system to deliver O2 (Levine, 2008, Taylor
et al., 1955).

With the Fick equation in mind, the first point at issue is to understand which adaptations
explain ET-induced increases in Qmax and how these specifically contribute to VO2max. This is
illustrated in Figure 2. In this regard, experimental studies have demonstrated that increases
in Qmax and VO2max following 6 weeks of ET are reverted to pre training levels after negating
training-induced gains in BV by means of phlebotomy (Montero et al., 2015b, Bonne et al.,
2014). This strongly suggests that early increases in Qmax and VO2max are exclusively
dependent on BV. Enhanced BV is thought to augment the pressure gradient from central
venous reservoir to right atrium leading to enhanced venous return, cardiac preload and
stroke volume via the Frank-Starling mechanism (Hopper et al., 1988, Coyle et al., 1986,
Convertino et al., 1991, Kanstrup and Ekblom, 1982). Of note, any BV-mediated increase in
Qmax has little influence on O2 delivery and thereby VO2max if only plasma volume (PV), but
not also the total red blood cell volume (RBCV) is augmented, since the O2 carrying capacity
of the blood is diminished (Montero et al., 2015c, Warburton et al., 2000, Keiser et al., 2015).
BV expansion induced by ET commonly comprises ~10 % increments in PV following a
single exercise bout and levelling off after approximatly 2 weeks, along with similar or lower
increments in RBCV noted after 6 to 12 weeks (Convertino, 2007, Sawka et al., 2000, Bonne
et al., 2014, Helgerud et al., 2007, Warburton et al., 2004, Montero et al., 2015b). With
longer-term ET, PV, RBCV and absolute hemoglobin mass have been shown substantially
enhanced (up to 40 %) in endurance athletes (Dill et al., 1974, Brotherhood et al., 1975,
Heinicke et al., 2001, Lundby and Robach, 2015). In these, donation of 1 unit of blood (450
ml) immediately prompts an 8 % decrease in VO2max, which is not re-established after 1 week
(Panebianco et al., 1995).

Endurance athletes also present cardiac eccentric hyperthropy, largely increased left
ventricular compliance and reduced total peripheral vascular resistance (i.e., decreased
afterload), all potentially facilitating higher stroke volume (Spence et al., 2011, Levine et al.,
1991, Fleg et al., 1994). Similar cardiac adaptations but of lesser magnitude are observed
after 6-12 months of ET in previously untrained individuals, although their independent
contribution to Qmax remains to be experimentally elucidated (Arbab-Zadeh et al., 2014,

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 Spence et al., 2011). Whilst speculative, cardiac adaptations might be partially the
consequence of chronically elevated BV and central venous pressure (CVP) (Convertino et
al., 1991), independently of direct ET effects. Taken together, it can be unequivocally
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concluded that increases in BV along with total oxygen-carrying capacity, as essentially
characterized by RBCV expansion, are fundamental for improving VO2max.

Despite the preponderant influence of RBCV on VO2max, there is some uncertainty regarding
the mechanisms stimulating erythropoiesis with ET. Acute (normoxic) exercise transiently
activates hypoxia inducible factor-2α in and erythropoietin (EPO) release from contracting
skeletal muscle (Lundby et al., 2006a, Rundqvist et al., 2009). Plasma EPO concentration
seems unaffected up to 48 h following 60 min of whole-body (running, cycling) exercise
(Bodary et al., 1999, Schmidt et al., 1991). In contrast, up to ~100 % increments in
circulating EPO levels have been reported immediately (Robach et al., 2014) (Schwandt et
al., 1991) and 2-3 days after (Robach et al., 2014, Schwandt et al., 1991, Roecker et al., 2006)
prolonged strenuous exercise ((ultra)marathon), although this is not a universal finding
(Weight et al., 1992). These observations could be confounded by concurrent changes in PV.
In addition, it has been proposed that the rapid increase in PV in the first few days of ET
might impel the erythropoietic system to approach a new equilibrium in that haematocrit is
partially restored to pre training levels (Jelkmann and Lundby, 2011, Sawka et al., 2000,
Schmidt and Prommer, 2008). Yet, there is no proof that augmented PV per se stimulates
erythropoietin (EPO) synthesis in the setting of normal RBCV. Moreover, the observation of
slightly higher increases in RBCV than PV after 6 weeks of ET suggests that RBCV
expansion might be, at least in part, not ultimately regulated by PV (Montero et al., 2015b).

Alternatively to hypoxia-related signals, the filling state of (and/or blood volume distribution
in) the cardiovascular system, as reflected by CVP, is markedly reduced during several hours
after whole-body exercise (Kirsch et al., 1975, Kirsch et al., 1986). This is detected by veno-
atrial and central arterial stretch receptors that stimulate the secretion of BV-regulating
hormones possibly contributing to the erythropoietic response (Montero et al., 2016). When
CVP is acutely decreased by whole-body tilting, plasma EPO concentration increases, an
effect mediated by concomitant increases in vasopressin, which may enhance EPO secretion
through the activation of V1a receptors (Engel and Pagel, 1995, Montero et al., 2016). Thus,
ET-induced erythropoiesis might well be independently regulated by parallel endocrine
feedback loops to those regulating PV and interstitial fluid homeostasis. Furthermore, ET
improves the hematopoietic microenvironment and alters the secretion pattern of

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 catecholamines, peptides (growth hormone, insulin-like growth factor) and steroid hormones
(testosterone, cortisol), all of which may influence red blood cell production and/or release
from the bone marrow (Hu and Lin, 2012). The relative importance of the above mechanisms
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in the erythropoietic response to ET remains elusive and a compelling challenge for
understanding the physiological bases of aerobic conditioning. Importantly, common
variants in genetic pathways that might modulate key physiological pathways controlling
convective O2 delivery have not been identified yet (Sarzynski et al., 2016). However, at
least on case report of a rare variant in a champion athlete associated with a very high RBCV
reinforces the crucial point raised herein (Thompson, 2012).

The contribution of adaptations associated with peripheral adaptations and associated O2

extraction to VO2max improvements merits attention. As previously mentioned, a-vO2diff is
found to be primarily increased with long-term ET, although phenotypic modifications
typically considered to enhance O2 extraction emerge in the early stages of ET (Montero et
al., 2015b). Herein, large increments in skeletal muscle capillarization (18 %) and
mitochondrial volume density (43 %) are observed after 6 weeks of ET but a-vO2diff is
unaltered (Montero et al., 2015b). In line with this, VO2max is not improved with 4-7 weeks
of one-legged ET when Qmax is not increased, even if maximal O2 extraction is enhanced in
the trained versus control leg (Gleser, 1973, Rud et al., 2012). Indeed, muscle O2 extraction is
not maximal at VO2max and recent experimental studies demonstrate a twofold functional
reserve in untrained individuals (Calbet et al., 2015).

One clever series of experiments in rats provides further insight into this fundamental
question. Davies and colleagues (Davies et al., 1982) studied iron-depleted animals who
were anemic and lacked normal mitochondrial function in their skeletal muscles. These
animals had reduced values for VO2max and endurance running time. Acute correction of their
anemia normalized their VO2max values but had little impact on their endurance running
time. This model confirms the primacy of convective O2 transport as the major determinant
of VO2max. In parallel with these observations are observations that in individuals who have
been training for many years, mitochondrial adaptations are uniformly high but dissociated
from VO2max (Lundby and Jacobs, 2016). In this line, in healthy humans mitochondrial
oxidative capacity exceeds that of O2 delivery at VO2max and hence is currently not considered
to limit O2 extraction (Boushel et al., 2011, Lundby and Montero, 2015). Collectively
considered, within-muscle adaptations enhancing maximal O2 extraction may therefore not be
crucial for increasing a-vO2diff and thereby VO2max. In contrast to our conclusions, opposite

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 viewpoints mainly based on theoretical models are available (Wagner, 1992, Wagner, 2015,
Lundby and Montero, 2015).
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O2 extraction (as measured) and a-vO2diff may be functions of the distribution of blood flow
among active/inactive muscle fibers and other tissues at distinct levels (whole-body, limb,
muscle) (Kalliokoski et al., 2005, Kalliokoski et al., 2001, Calbet et al., 2006a). Blood flow
distribution (BFD) during exercise is plausibly determined by the interplay of vascular
dilator/constrictor function, sympathetic drive and microvascular structure (Calbet et al.,
2006a, Lundby et al., 2008, Emerson and Segal, 1997, Joyner and Casey, 2015). A change in
BFD could explain the observation that some individuals exhibit no change in leg a-vO2diff
but increased whole-body a-vO2diff after 12 weeks of ET with cycle ergometry (Beere et al.,
1999a). Moreover, BFD is improved and a-vO2diff augmented in leg exercising muscle
(quadriceps femoris) during submaximal exercise in chronically trained individuals
(Kalliokoski et al., 2001). Regardless, the fact that a-vO2diff is near peak levels at exhaustion
in un- and trained individuals precludes a major impact on VO2max of any potential
improvement in BFD during (sub)maximal exercise (Lundby et al., 2006b, Rud et al., 2012).

Non-responders to exercise training: our concerns.

In recent years much attention has been given to the emerging concept that some individuals
seemingly do not respond to endurance exercise training with a marked improvement in
VO2max. These individuals have accordingly been termed non-responders. Considering the
immense health benefits associated with cardiorespiratory fitness and improvements in
VO2max, and the general relationship between fitness and all-cause mortality, it is possible
that such findings may lead certain individuals to refrain from exercise training due to
uncertain benefits and even what some have called adverse responses to training for selected
risk factors (Bouchard et al., 2012). In this context we are also concerned that the underlying
scientific evidence for the non-responder phenomenon has underappreciated limitations, and
that a hypothesis neutral search for DNA variants that determine responses to training is
unlikely to yield explanations that can be reconciled with known physiological principles
related to oxygen transport and gas exchange that we have outlined above. We are also
concerned that the non-responder concept fails to acknowledge the protective effects of
exercise that go beyond changes in traditional risk factors associated with the endurance
trained state (Joyner and Green, 2009).

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 Where did the non-responder concept come from?

The term non-responders was coined based on data gathered from the Heritage Family Study.
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In this landmark study 483 sedentary individuals as part of family groups were assigned to 20
weeks of exercise training three times a week. Initially they trained for 30 min at a heart rate
corresponding to 55% of their VO2max, but every two weeks the intensity and duration was
progressively increased until they exercised 50 min at 75% of VO2max. This was achieved in
training week 14. The range of improvements in VO2max varied from zero to more than 1l
O2.min-1 (Bouchard et al., 2011, Skinner et al., 2001). Subsequent retrospective analysis
suggested that up to 49% of the variation in the response to training could be ascribed to
heredity based on the cumulative presence or absence of 21 single nucleotide polymorphisms.
These results are however based on stepwise regression procedures thoroughly discredited in
the literature and thus should be interpreted with caution (Mundry and Nunn, 2009).
Additionally, the number of gene variants that might be associated with increased VO2max is
increasing in number and as of 2009 “The human gene map for performance and health-
related fitness phenotypes” included 239 genes (Bray et al., 2009). However most of these
variants or pathways are not clearly linked to the physiological systems responsible for the
bulk transport of oxygen from air to tissues that dominate the physiological determinants of
VO2max and almost all have very small effect sizes. In this context, the Heritage author’s
have acknowledged that candidate gene and genome wide linkage studies have failed to
clearly contribute to our understanding of the molecular basis for variations in exercise
adaptations (Sarzynski et al., 2016). It is also interesting to note that variants of the
angiotensin converting enzyme gene once thought to be associated with very high levels of
VO2max were not confirmed when a large sample of elite endurance athletes were studied
(Rankinen et al., 2000). We highlight this finding because it shows that even for a gene that
might plausibly play a role the regulation of blood volume and/or cardiac hypertrophy a clear
story about DNA variants and VO2max is hard to come by.

An often ignored or overlooked finding from the Heritage study is that exercise intensity was
automatically controlled by heart rate monitoring. This may have caused training workloads
to be gradually reduced over the course of a training session as heart rate drifts upwards with
continuous exercise. Indeed, fluctuations in training workload did occur and partly explained
the training response (Sarzynski et al., 2016). While not specifically reported, the non-
responders might reasonably be the individuals with the greatest mis-match between target
and achieved workloads, which would beg the obvious question: Did the non-responders

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 simply not respond to the training regime as the intensity and/or volume was too little? It
should also be noted that smaller studies in a number of cohorts including identical twins
(Prud'homme et al., 1984) showed highly variable changes in VO2 max in response to what
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might be described as the adult fitness style training outlined above for the Heritage study.
Finally, while it is easy to be critical of Heritage in retrospect, it should be remembered that
this was an absolutely state of the art study when it was developed and required a truly
impressive degree of coordination and intellectual insight to design and execute it also
included women along with several racial groups and a range of age. That a clear pattern of
DNA variants did not emerge to explain a high percentage of the Heritage findings likely
represent limitations that have emerged subsequently in what has been termed the “common
variant” hypothesis which was widely anticipated at the time the study was conceived
(Shields, 2011, Weiss, 2008).

Will all previously untrained individuals gain from exercise training if pushed sufficiently?

At the turn of the last century (e.g. 1900) it was suggested that exercise training facilitates
VO2max in humans and that both volume and intensity may influence the outcome hereof.
Soon after and based on experiences of athletes and coaches high intensity repetition, fartlek
and interval training was widely adopted and revolutionized running performances. Two of
the most notable athletes to employ such approaches into their training regimes were Roger
Bannister (first person under 4:00 min for the Mile) and Emil Zatopek (Multiple Olympic).
Some of the earliest scientific studies of athletes who used high intensity training showed that
with even modest volumes of high intensity training very high VO2max values could be
achieved (Robinson et al., 1937). Additionally, early work from a number of labs provided
evidence that these high values for VO2max were associated with evidence for very large
stroke volumes and total body hemoglobin (Kjellberg et al., 1949a, Kjellberg et al., 1949b)

Since then a myriad of scientific studies have demonstrated that interval training is effective
in increasing VO2max. For example, after two months of intense interval training (15 sec
maximum running/ 15 sec rest in the end totaling 15 min of high intensity exercise or 3 min
maximum running/ 3 min rest, again performed so that total time of high intensity exercise =
15 min) conducted three times per week (i.e. same as in the Heritage study) “all subjects
(n=37) demonstrated increases in this (VO2max) functional capacity” (Figures 2 and 3 in
(Knuttgen et al., 1973)). Later Hickson and co-workers (Hickson et al., 1977) found that a 10

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 week long training intervention alternating between daily (6/week) interval (5 × 5 min at
VO2max) and continuous (as fast as possible for 30-40 min) running improved VO2max by at
least 700 ml O2.min-1 in all (n=8) participants. These training regimes are obviously of much
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more rigorous nature than that applied in the Heritage Family Study and one could be
tempted to conclude along the lines of the conventional no pain – no gain wisdom. It can also
be argued however that not many individuals, and perhaps especially untrained individuals
will be willing to endure such trainings. The studies do however suggest that with the right
training that non-responders to exercise training do not exist. In further support, a recent
meta-analysis (Bacon et al., 2013) revealed that interval/high intensity training improves
VO2max slightly greater than those typically reported with “adult fitness based continuous
training” despite many of the interval studies were of shorter duration and with fewer training
sessions per week. Ross and colleagues (Ross et al., 2015) performed a much needed study
demonstrating the importance of exercise intensity and volume in regards to VO2max
improvements. In that study 192 individuals were assigned to 24 weeks of training consisting
of 5 sessions/week including either A) 180/300 kcal (female/male) conducted at 50% of
VO2peak, B) 360/600 kcal conducted at 50% or C) 360/600 kcal conducted at 75%. In groups
A and B 38.5 and 17.6%, respectively, of the participants were classified as non-responders
after the training intervention, whereas the number was 0 in group C. This study thus
demonstrates that when doubling training volume (group A vs B) then non-responders are
decreased by 50%, and if more intensity is added to that (group B vs C) then the phenomenon
entirely disappear. Based on this study it could be speculated that if the participants in the
Heritage study had trained 5 sessions per week rather than 3, then perhaps the outcome may
have been entirely different.

How should one then train to avoid ending up as a non-responder? Nordesjö (Nordesjö, 1974)
published similar improvements in VO2max (with improvements noted in all participants)
between groups running either A) 15 min once per week at a heart rate (HR) of 190
beats.min-1, B) 3 × 60 min runs/week at a HR of 150 or C) 5 × 120 min runs/week at HR of
110. Thus an obvious tradeoff between intensity and volume exists, and it seems likely that if
the training intensity had been higher in the low intensity group (Group A) in the study by
Ross (Ross et al., 2015) and also in the Heritage study then the rate of non-responders would
be minimal or non-existent. There are also number anecdotal observations (Astrand and
Rodahl, 1986) on “average” young men who have subjected themselves to prolonged and
intense almost professional style training and achieved VO2 max values of nearly 60 ml.kg-

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 1
.min-1. While this value seems high at first gland imagine a young man with a VO2max of
3.2 l.min-1 and body weight of 80 kg. Such an individual would almost certainly be able to
increase his max to roughly 4 l.min-1 with several years of training. If at the same time his
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body weight dropped to 70 kg his VO2max would equal 57 ml.kg-1.min-1. By no means elite,
but a value sufficient perhaps to run a marathon in just over three hours. Such values
certainly do not seem inconceivable based on the original study by Hickson and colleagues
and more recent studies by Howden and colleagues (Howden et al., 2015). In the later study it
was surprisingly demonstrated that females respond somewhat less to a training intervention
as compared to males. The very small number of participants in that study (7 males and 5
females) urges for more studies on this topic including greater subject numbers and the
concomitant assessment of hemoglobin mass. Furthermore, it is very important to determine
iron status of the included females as ~20% may be anemic and hence likely less prone to
expand their hemoglobin mass. Additionally, much of the data we have focused on was
obtained from studies in men because only limited numbers of women were studied before
the 1990s or 2000s. Finally, will any genetic markers associated with responses to short term
adult fitness training be replicated in response to more prolonged and intense endurance
training?

Conclusion and perspective

We are of the opinion that overwhelming data exists demonstrating that all previously
untrained humans will respond to endurance exercise training regimes provided the stimulus
is sufficient. These improvements are for the greatest part associated with improvements in
RBCV observed within weeks, leading to higher O2 carrying capacity, stroke volume and
hence O2 transport capacity. Surprisingly little is known regarding the mechanisms
facilitating the expansion of RBCV with exercise training. With months of training, stroke
volume could also be facilitated by cardiac growth and moderately increased ventricular
compliance. Considering that factors facilitating VO2max with endurance training are largely
limited to the expansion of RBCV and stroke volume, it might make sense for studies aiming
to predict or explain the biological mechanisms responsible for variations in VO2max
trainability should: 1) use a training program designed to elicit maximum increases in VO2max
within a given individual. This means that prolonged and intense training will be required,
and 2) focus on hormonal responses, gene variants and molecular adaptations of relevance for

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 the dominant physiological pathways we have emphasized in this paper. From an
evolutionary perspective, when DNA variants are found in specific populations (such as
groups that have been exposed for many generations to profound hypoxia), there is
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substantial evidence for selection and relatively common variants in key physiological
pathways emerge (Simonson, 2015). In this context, the estimated VO2max values for male
hunter gatherers are on the order of 55-60 ml.kg-1.min-1 (Joyner and Casey, 2015). Since
these values are similar to those attainable by most lean active young males, we question – in
the absence of a clear argument and evidence for selection pressure - whether easily
identifiable DNA variants in the key physiological pathways for VO2max we have outlined
will be identified for both average individuals and also elite endurance athletes.

Conflict of Interest: The authors have no conflict of interests to declare.

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Figure 1. Correlations between VO2max, maximal cardiac output (A) and red blood cell
volume (B). Some of these data points are previously published (Montero et al., 2015a,
Lundby and Robach, 2015). All data were obtained within the “Lundby lab” by means of CO
(red blood cell volume) and inert gas (cardiac output) re-breathing.

Figure 2. Physiological adaptations underlying improvements in maximal oxygen

uptake (VO2max) with exercise training

Expansion of plasma volume is observed within hours following whole-body exercise and
remains elevated with regular exercise training (Convertino, 2007, Sawka et al., 2000, Bonne
et al., 2014, Helgerud et al., 2007, Warburton et al., 2004, Montero et al., 2015b). Increases in
red cell blood volume and total oxygen-carrying capacity ensue after few weeks of training
(Montero et al., 2015b, Bonne et al., 2014). The resultant augmentation of blood volume
facilitates venous return leading to higher end-diastolic volume and stroke volume (SV) via
the Frank-Starling mechanism (Hopper et al., 1988, Coyle et al., 1986, Convertino et al.,
1991, Kanstrup and Ekblom, 1982). Changes in plasma and red blood cell volumes will also
affect the a-vO2diff. Months of training may result in cardiac eccentric hyperthropy,
moderately enhanced ventricular compliance and reduced afterload, possibly facilitating
higher end-diastolic volume (Spence et al., 2011, Levine et al., 1991, Fleg et al., 1994).
Maximal heart rate (HR) is commonly not affected by exercise training. Skeletal muscles
adaptations potentially contributing to O2 extraction and thereby arteriovenous oxygen
difference (a-vO2diff) mainly include increases in mitochondrial volume density/oxidative
capacity and capillarization. These are clearly noticed in the early weeks of training but have
no influence a-vO2diff (Montero et al., 2015b), plausibly attributable to the twofold functional

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 reserve in muscle O2 extraction at VO2max in the untrained state (Calbet et al., 2015).
Enhanced a-vO2diff is evident following approximately 12 weeks of training (Beere et al.,
1999a, Montero and Diaz-Canestro, 2015, Montero et al., 2015b). This could be due to
Accepted Article
improved blood flow distribution primarily determined by combined adaptations in vascular
dilator/constrictor function and microvascular structure (Calbet et al., 2006a, Lundby et al.,
2008, Emerson and Segal, 1997). Ultimately, there is little ‘room’ for increasing a-vO2diff and
thus VO2max improvements are essentially determined by increases in SV along with
relatively preserved oxygen-carrying capacity of the blood.

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Accepted Article

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