Near-infrared spectroscopy andskeletal muscle oxidative function invivo in health and disease: a review
from an exercise physiology
perspective
Bruno Grassi
Valentina Quaresima

Bruno Grassi, Valentina Quaresima, “Near-infrared spectroscopy and skeletal muscle oxidative function invivo in health and disease: a review from an exercise physiology perspective,” J. Biomed. Opt.
21(9), 091313 (2016), doi: 10.1117/1.JBO.21.9.091313.

Journal of Biomedical Optics 21(9), 091313 (September 2016)

REVIEW

Near-infrared spectroscopy and skeletal muscle
oxidative function in vivo in health and disease:a review from an exercise physiology perspective
Bruno Grassia,b,* and Valentina Quaresimaca

University of Udine, Department of Medical and Biological Sciences, Piazzale M. Kolbe 4, Udine, I–33100 Udine, Italy
Institute of Bioimaging and Molecular Physiology, National Research Council, I-20030 Segrate, Milan, Italyc
University of L’Aquila; Department of Life, Health and Environmental Sciences, Via Vetoio, I-67100 L’Aquila, Italyb

Abstract. In most daily activities related to work or leisure, the energy for muscle work substantially comes fromoxidative metabolism. Functional limitations or impairments of this metabolism can significantly affect exercise
tolerance and performance. As a method for the functional evaluation of skeletal muscle oxidative metabolism,
near-infrared spectroscopy (NIRS) has important strengths but also several limitations, some of which have been
overcome by recent technological developments. Skeletal muscle fractional O2 extraction, the main variablewhich can be noninvasively evaluated by NIRS, is the result of the dynamic balance between O2 utilization
and O2 delivery; it can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise performance and exercise tolerance in healthy subjects (in normal and inaltered environmental conditions) and in patients. In the right hands, NIRS can offer insights into the physiological and pathophysiological adaptations to conditions of increased O2 needs that involve, in an integrated
manner, different organs and systems of the body. In terms of patient evaluation, NIRS allows determinationof the evolution of the functional impairments, to identify their correlations with clinical symptoms, to evaluate theeffects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights. © 2016
Society of Photo-Optical Instrumentation Engineers (SPIE) [DOI: 10.1117/1.JBO.21.9.091313]

Keywords: near-infrared spectroscopy; skeletal muscle bioenergetics; skeletal muscle oxidative metabolism; exercise physiology;
exercise tolerance; review.
Paper 150870SSVR received Jan. 6, 2016; accepted for publication Jun. 28, 2016; published online Jul. 21, 2016.

1

Introduction

Near-infrared (NIR) spectroscopy (NIRS) applied to skeletal
muscle has often been seen with some skepticism by many
exercise physiologists. Once at a scientific meeting, one of
the authors of the present review was told by Dr. Bengt
Saltin (one of the most influential exercise physiologistsof the last decades): “Unless you manage to measure O2 consumption, this technique has not much physiological interest.”
We respectfully disagree with Dr. Saltin’s statement.
Although we recognize that NIRS has several limitations,some of which have been overcome by recent technologicaldevelopments, the method also has important strengths andcan give valuable (and noninvasive) functional insights into
skeletal muscle oxidative metabolism in vivo during exercise,in health and disease.
At this purpose, the present review has been devoted, froman “exercise physiology point of view,” to discuss some of themain issues related with the role of NIRS in the functional
assessment of oxidative metabolism in skeletal muscles during
exercise, with specific attention to integrative aspects and to thefactors limiting exercise tolerance. Attention will also be paid
to studies carried out in diseased populations, as well as to*Address correspondence to: Bruno Grassi, E-mail: bruno.grassi@uniud.it

Journal of Biomedical Optics

some recent and exciting technical and methodologicaldevelopments.

2

Oxidative Metabolism During Exercise
and Tools of Investigation

During supramaximal exercise lasting up to a few seconds, themetabolic power output in humans can increase by about 150 to
200 times compared with the value observed at rest. Among thetissues of the body, only skeletal muscle can sustain such
extraordinary increases in metabolic power output, which ismade possible by the splitting of high energy phosphates present
in muscle as adenosine triphosphate (ATP) and creatine phosphate (PCr). If the exercise is carried out for longer periodsof time (up to several minutes, or even hours), the maximal metabolic power output, which can be sustained, decreases hyperbolically as a function of the duration of the exercise1–4 andoxidative metabolism becomes the prevalent, or substantiallythe only, mechanism responsible for ATP resynthesis. In
other words, in most of our daily activities, either related towork or leisure, the energy for muscle work substantiallycomes from the oxidation of glucose and lipids in muscle fibers,
culminating in oxidative phosphorylation at the mitochondrial
respiratory chain.
It is not surprising, then, that the maximal power by oxidative
metabolism and the fraction of this power, which can be sustained for relatively long periods of time, are intrinsically related
to exercise performance and tolerance. An impairment of

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oxidative metabolism, which occurs in several types of patients,
inevitably leads to a reduced exercise tolerance, which may represent one of the main determinants of the clinical picture andquality of life, as well as an important predictor of mortality.5
The availability of reliable tools to investigate in vivo, with
precision and reproducibility, muscle oxidative metabolism,
possibly noninvasively and with a reasonably elevated temporal
resolution, appears therefore of utmost importance. This applies
to athletes (sports activities), healthy subjects (both in normal
and in altered environmental conditions), and patients affectedby many diseases. In the patients, these tools would allow thefollow-up of the impairments with time, the identification ofpotential correlations with clinical symptoms, the evaluationof the effects of therapeutic or rehabilitative interventions,and could also yield pathophysiological and diagnostic insights.
_ 2 ) can be measured
Whole body oxygen consumption (VOwith relatively good precision (variability of measurements ofabout 5% in the best laboratories) at the mouth of the subject_ 2, VO_ 2p ) by the classic
or at the alveolar level6 (pulmonary VO
open circuit method. These measurements have been carried out
in innumerable studies and lead to the definition of concepts like
_ 2p;max ), which evaluates the intethe maximal aerobic power (VOgrated performance of the respiratory, cardiovascular, and skeletal muscle components of the O2 pathway from ambient air
to the mitochondria of skeletal muscles. The availability of_ 2p with an elevated temporal resolution
methods to determine VO(“breath-by-breath” measurements)6–8 expanded the spectrum of
the functional evaluation variables and allowed the introductionof concepts such as the “gas exchange threshold,”9 the “critical_ 2 “kinetics.”3,4,6,10 The
power,”1 or the various phases of the VO_
main limitation of the whole body VO2p measurements resides
in the impossibility to discriminate between the exercisingmuscles and the rest of the body, as well as between different
muscles engaged in the exercise. Moreover, the presence of O2
stores between the site of measurement (the mouth or thealveoli) and the sites of gas exchange at the skeletal muscle
level complicates data interpretation during metabolictransitions.6 A further limitation derives from the diffusion of
automated and apparently easy-to-use “metabolimeters,”
which led to the proliferation of data and studies of dubious
quality.
_ 2 , blood flow and O2 extraction
The relationship between VO
can be expressed by the Fick equation, based on the principle ofmass conservation. If applied “across” a single muscle or a single muscle group, the equation reads as follows:_ m  ½Cða-vÞO2m ;_ 2m ¼ Q
VO

EQ-TARGET;temp:intralink-;e001;63;258(1)_ m muscle blood flow,_ 2m represents muscle VO_ 2, Qin which VO
and Cða-vÞO2m arterial-venous O2 concentration difference
across the muscle.
By rearranging Eq. (1):_ m:
_ 2m ∕Q
Cða-vÞO2m ¼ VO

EQ-TARGET;temp:intralink-;e002;63;181

3Near-Infrared Spectroscopy from an
Exercise Physiology Point of View

Several excellent review articles about the application of NIRS
in skeletal muscle have been published.18–25 These reviews carry
the relevant information on principles, methods, instruments,and so on. Ferrari et al.24 included a useful list of practical recommendations. The present review article has been specifically
devoted to discuss, from an “exercise physiology point of view,”
some of the main issues related with the role of NIRS in thefunctional evaluation in vivo of oxidative metabolism in skeletal
muscles, within an integrated perspective related to exercise tolerance. A specific attention will be paid to NIRS studies carriedout in diseased populations, as well as to some recent and exciting technical and methodological developments. The latter testify how NIRS, as a tool of functional investigation of skeletal
muscles, is still in a steep portion of a gain in knowledge versus
time curve. The role of NIRS in evaluating oxidative skeletal
muscle performance in sport activities and in athletes, which
would deserve a dedicated review article, is not covered in
the present article. Likewise, the effects of exercise on brain
hemodynamics, as measured by NIRS,26,27 as well as the roleof NIRS in the evaluation of brain function21,25 are notdiscussed.

3.1

(2)

_ 2m measurements have been carried out with a high temporalVO
resolution also during metabolic transitions in humans11,12 or in
isolated in situ animal models.13,14 The main limitations of thisapproach can be summarized as follows: invasiveness of themeasurements; technical problems inherently associated within vivo blood flow measurements; the substantial impossibility(in human studies) to sample venous blood directly effluent
Journal of Biomedical Optics

from the exercising muscle, with a resulting “contamination”
of venous blood coming from other muscles.
Alternative methods allowing a functional evaluation in vivoof skeletal muscle oxidative metabolism derive from the utilizations of phosphorus (31 P) or proton (1 H) nuclear magnetic resonance spectroscopy (31 P-MRS, and 1 H-MRS, respectively). As
far as 31 P-MRS, the method has been utilized to determine thekinetics of recovery of muscle [PCr] following exercise. After
assuming the equilibrium of the creatine kinase (CK) reaction,
the rate of recovery of [PCr] is a function of mitochondrial ATP
production, and therefore it can be considered a tool for evaluating the function of skeletal muscle oxidative metabolism.15
1 H-MRS, on the other hand, detects myoglobin (Mb) desaturation, thereby allowing an estimation of intracellular PO2.16,17
These technologically formidable methods are intrinsically limited by the very high cost of the instrumentation and by the strict
constraints on the exercise modality determined by the size ofthe bore of the magnet.
In short, the above mentioned methods, although widely utilized, have intrinsic limitations. Therefore, noninvasive, precise,
reproducible, and relatively low-cost functional evaluation tools
to be utilized in vivo, characterized by an elevated temporal
resolution, allowing the discrimination between (and possibly
within) skeletal muscle groups, would be needed. Does NIRS
satisfy, at least in part, these needs? The following sectionsof the present review will deal with this issue.

Tissue Interrogated by the Near-Infrared
Spectroscopy Probe

When the probe is applied on the skin overlying a muscle ofinterest, NIRS instruments can interrogate only a relatively
small (2 to 6 cm3 ) and superficial volume of skeletal muscle
tissue.28 It is generally accepted that the depth of penetrationof the NIR light in tissues roughly corresponds to half of the
distance between the light source and the detector (which isusually between 3 and 5 cm). This intrinsic technical limitation
makes the measurement of the thickness of the skin and

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∼20 mm would presumably make NIRS measurements, as carried out by standard instruments, rather meaningless in terms ofinvestigating skeletal muscle. This precludes the utilization of
the method in subjects with a relatively thick layer of subcutaneous fat, such as obese patients or in patients with significant
muscle atrophy.

subcutaneous adipose tissue layer, at the site of placement of the
NIRS probe, mandatory. The measurement can be carried out by
a caliper or, more precisely, by ultrasound.29,30 The recent availability of low-cost hand-held ultrasound devices has significantly facilitated these measurements. Although no cutoff
values are recognized as standard, a value greater than

Table 1 Main commercial muscle NIRS oximeters.

Company,
country

Technique

Oxymon Mk IIIa

Artinis, The
Netherlands

Exercise Monitor
Hb11b

Astem,Japan

Timeresolution No. of Measurable(Hz)
channels parameters Approvals

Type ofsourcesN. ofwl

Multidistance
CW

laser

2

250

2

SO2m

www.artinis.com

Multidistance
CW

LED

2

1

1

SO2m

www.astem-jp.com

Multidistance
CW

LED

5

0.5

4

SO2m

FDA,
CE

www.casmed.com

NIRO-200 NXa

Hamamatsu, Multidistance
Japan
CW

LED

3

20

2

SO2m

CE

www.hamamatsu.com

tNIRS-1a

Hamamatsu, TD
Japan

laser

3

0.2

2

SO2m , atHb

CE

www.hamamatsu.com

OxiplexTS™aISS, UnitedStatesMultidistance
FD

laser

2

50

2

SO2m , atHb

O3™ Regional
Oximetrya

Masimo,UnitedStatesMultidistance
CW

LED

4

1

up to 6

SO2m ,
SO2p

CE

www.masimo.com

Multidistance
CW

LED

2

0.2

up to 4

SO2m

FDA

www.medtronic.com

moorVMS-NIRS

Moor, United Multidistance
Kingdom
CW

LED

2

5

2

SO2m

CE

www.moor.co.uk

NIMOabNirox, Italy

Multidistance
CW

laser

3

40

up to 4SO2m

CE

www.nirox.it

SenSmart™
Model X-100a

Nonin,
UnitedStatesMultidistance
CW

LED

4

0.6

up to 5

SO2m , SO2p

FDA

www.nonin.com

BOM-L1 TR
SFOmegawave,
Japan

Singledistance

laser

3

1

1

SO2m

Cerox™ 3210Fa

Ornim, Inc.,
UnitedStatesMultidistance
CW

laser

3

0.33

up to 4

SO2m , flowc

FDA

www.ornim.com

CareGuide™
4100

Reflectance, MultidistanceMed., United CWStatesLED

Multiple

0.03

1SO2m , pHm

FDA

www.reflectancemedical.
com

DeviceFore-sight Elite®a CAS, Inc.,
UnitedStatesINVOSTM 5100Ca Medtronic,
UnitedStatesWebsiteswww.iss.com

www.omegawave.co.jp

Note: atHb, absolute total hemoglobin concentration; CE, European conformity marking as a medical device; CW, continuous-wave spectroscopy;
FD, frequency-domain spectroscopy; FDA, Food and Drug Administration’s approval; LED, light emitting diodes; N., number; pHm, muscle pH;
SO2m , muscle oxyhemoglobin saturation; SO2p , peripheral arterial oxyhemoglobin saturation (measured by pulse oximetry); TD, time-domain spectroscopy; wl, wavelengths.
aBrain oximeter utilized also in muscle studiesb
Oximeter with fat-layer compensationc
Cortical microcirculation blood flow measurement using Doppler shift in coherent light signals.

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The fact that the NIR light has to cross the skin and subcutaneous fat in order to reach the underlying skeletal muscle tissue
inevitably causes a sensitivity problem and represents a “contamination” of the signal of interest, which is the one comingfrom skeletal muscle. The problem can be particularly significant in conditions in which skin blood flow increases during
exercise for thermoregulatory purposes (see also below).
Several “correction algorithms” have been proposed (see thediscussion in Ferrari et al.24), which, however, are not included
in most of the commercial instruments (Tables 1 and 2). Ohmae
et al.31 have recently described a simple method, which does not
require ultrasound, for the sensitivity correction for the influenceof fat thickness on muscle oxygenation, based on the estimationof fat thickness by time-domain NIRS (TD-NIRS) (see below).
van Beekvelt et al.32 and Koga et al.29 proposed other correction_ 2m [estimatedalgorithms based on the assumption that resting VO
on the basis of the concentration increases in deoxy-hemoglobin(Hb) and deoxy-myoglobin (Mb), [deoxy(Hb+Mb)]) after applying a cuff occluding the arterial circulation, see below] would beinversely correlated with skin + subcutaneous tissue thickness.
Another approach, based on [total(Hb+Mb)] values obtained at
rest by quantitative multichannel TD-NIRS, has been proposed.33
In the quadriceps muscle (often investigated by NIRS studies)and in other muscles as well, deeper muscle regions are characterized by a greater proportion of oxidative fibers compared tosuperficial regions34 and by a more oxidative energy metabolism.
Accordingly, the deeper fibers have a greater sensitivity towardvasodilatory control mechanisms.35 An anatomical “gradient”
in fiber type is presumably associated with a gradient also interms of metabolism and blood perfusion.36 This has been substantially confirmed, in the quadriceps muscle of humans, instudies carried out by utilizing a “high-power” TD-NIRS instrument,37,38 which allows interrogation of slightly deeper portionsof muscle. This instrument, which delivers NIR light with a power
about 30 times higher than that of traditional NIRS instruments,
allows an effective mean penetration depth of about 3 cm and may
represent a substantial technical improvement.
Another problem related to the small volume of tissue interrogated by conventional NIRS instruments derives from the factthat muscle activation and metabolism,39 and even more markedly so blood perfusion36,40,41 are heterogeneously distributed
within and between exercising muscles. Excellent reviews on
metabolic and blood flow heterogeneities in skeletal muscle,in health and disease, have been recently published.42,43 Sinceskeletal muscle fractional O2 extraction (see below) can increaseduring exercise only three to four times above the values at rest,_ 2m can increase 100-fold, directing blood flowwhereas VO
according to O2 needs is crucial to allow sustained contractile
performance.43
Most studies (but not all44) found good matching between_ 2m ¼ Q_ m  CaO2 , in which CaO2 repmuscle O2 delivery (QO_ 2mresents the arterial O2 content), or substrates delivery, and VO
between macroscopic (volume of several cm3 ) regions of amuscle, or between different muscles involved in theexercise.30,45,46 However, when the resolution capacity of themeasurements went down to about 1 cm3 , the spatial matching_ 2m and VO_ 2m (assessed via PCr depletion) was far
between QO
from being perfect.45 The presence (or absence) of a matching
between the two variables could be more functionally relevant ata microscopic level. According to Segal,47 the recruitment ofcontiguous “microvascular units,” whose volume is orders ofmagnitude smaller than the volume investigated by NIRS,
could be difficult to match with the recruitment of motor
units, whose fibers are sparse within the muscle. In otherwords, blood flow cannot specifically increase to a specificmuscle fiber or to the fibers of a motor unit,47 but must rather

Table 2 Main commercial continuous wave portable/wearable NIRS systems for muscle studies with wireless data transmission.

DeviceCompany, countryaTechnique

N. of
wl

Timeresolution(Hz)

No. ofchannels

Measurable
parameters

Websites

OctaMonArtinis, The Netherlands

Singledistance

2

50

8

O2 Hb∕HHb
changeswww.artinis.com

PortaMon Mk IIb

Artinis, The Netherlands

Multidistance

2

10

1

SO2m

www.artinis.com

BSX Insight 2.0

BSX Athletics, LLC,
United States

n.a.

n.a.

n.a.

1

SO2m , LT

www.bsxinsight.com

PocketNIRS
Duo®c,dDynaSense Inc., Japan

Singledistance

3

60

2

O2 Hb∕HHb
changeswww.dynasense.co.jp

Moxye

Moxy Monitor,
United States

Monte Carlomodeling

4

2

1

SO2m

www.moxymonitor.
com

MyNimoNirox, Italy

Multidistance

3

10

1

SO2m , atHb

www.nirox.it

OxiTor-M2

Pathonix Innovation
Inc., Canada

Multidistance

2

30

1

SO2m

www.pathonix.com

Note: atHb, absolute total hemoglobin concentration; HHb, deoxyhemoglobin; LT, lactate threshold evaluation by SO2m tracing; n.a., not available;N., number; O2 Hb, oxyhemoglobin; SO2m , muscle oxyhemoglobin saturation; wl, wavelengths.
aMuscle imager.
b
Accelerometer is available on request.
c
Commercially available only in Japan.
dSmartphone controllable system.
e
Water resistant case.

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increase over a relatively wide region of the muscle, leading to
the possibility of “over-perfusing” inactive or relatively inactive
fibers.
_ 2m
In any case, the apparently good matching between QO_ 2m in different macroscopic areas of the same muscle,
and VOor of different muscles,45,46,48 appears to be difficult to reconcile
with the macroscopic heterogeneity of muscle deoxygenation
observed by several studies.29,37,49 The issue, in our opinion,
is not settled, and further studies with higher spatial resolution
devices are needed.
In summary, the area of muscle investigated by the NIRS
probe may not represent,in terms of fiber types, fiber activation:_ 2m , a reliable picture of theand the matching of VO2m and QO
situation in the whole muscle. In this respect, however, it should
be considered that the same limitation, frequently overlooked, isintrinsically associated with other diffusely utilized methods,
which investigate only a small and relatively superficial portionof a muscle, such as muscle biopsy.

3.2

Where do the Near-Infrared Spectroscopy
Signals Come From?

The NIRS signals are the result of the weighted average of theO2 saturations of the heme groups of Hb in the vascular bed(small arteries, arterioles, capillaries, venules, small veins)and of the Mb heme group in muscle fibers. In terms of Hb,
most of the signal comes from small vessels, because larger
arteries and veins (greater than ∼1 mm in diameter) havevery high heme concentrations, which absorb all the NIR
light. Although it is often considered that venous–venular compartments represent the majority of blood present in skeletal
muscle,19,20 direct measurements suggest that this may not be
the case. According to Poole and Mathieu-Costello,50 capillaries
would contribute to >90% of the total blood volume in muscle.
According to these data, the storage of blood in veins would
mainly occur externally to the muscle. Thus, the vascular component of the NIRS signals would predominantly come from thecapillaries. Since in normal conditions, all regions of the muscle
receive nearly-fully oxygenated arterial blood, oxygenation
changes detected by NIRS would mainly reflect changes incapillary (Hb-related) and intracellular (Mb-related) O2 levels.
A relative controversy exists about the role played in NIRS
signals by the heme group in Mb. It has been traditionallyassumed that Hb is responsible for most of the overall NIRS
signal.51,52 The concept is supported by the observation of strong
correlations, observed in different experimental models,
between muscle oxygenation values obtained by NIRS andO2 saturation in venous blood (see below). Studies carriedout by NIRS in combination with 1 H-MRS, aimed at distinguishing between Hb and Mb desaturation during ischemia
and exercise, yielded conflicting results.53,54 According to modeling papers,55,56 Mb may contribute to ≥50% of the total[Hb+Mb] NIRS signal. This concept is supported by a study
in which the contribution of Hb and Mb to in vivo optical spectra
was determined by wavelength shift analysis.57
The relative role of Hb versus Mb is likely different at restand during exercise. As discussed above, most of the intravascular NIRS signals likely come from the capillaries. In resting
conditions, capillary hematocrit can be significantly lower thanthat in the systemic circulation.58 During exercise, on the other
hand, capillary hematocrit increases, reaching values that are notsubstantially different from those in larger vessels.58 Thus, theJournal of Biomedical Optics

role of Hb (versus that of Mb) in determining the NIRS signals
would be higher during exercise compared to rest.
The possibility to separate Hb and Mb desaturation could
give valuable insights into the mechanisms regulating peripheral_ 2m . For example, by applying NIRS on a
O2 diffusion and VO
rat model of isolated hindlimb, perfused with Hb-free welloxygenated Krebs-Henseleit buffer, Takakura et al.59 isolated
the signal deriving from Mb desaturation and could calculate
the intracellular PO2, making inferences on the role of Mb inO2 supply to mitochondria at exercise onset.
In any case, the whole diffusion pathway, including vascular
Hb and intracellular Mb, would desaturate during exercise witha similar time course.56 Therefore, a greater contribution of Mb
than Hb to the NIRS [deoxy(Hb+Mb)] signal would not invalidate the interpretation that changes in this signal reflect frac_ 2m andtional O2 extraction, the dynamic balance between VO_ 2m in the volume of tissue under consideration.56 This
QO
would apply even if a separation between the Hb and the Mb
signal is not feasible or is not carried out. In the next sectionsof the manuscript, we will discuss why, in our opinion, fractional
O2 extraction is by itself of interest.
In terms of the intracellular signals detectable by NIRS, cytochrome oxidase can also absorb NIR light. However, there issubstantial agreement that assessment of the redox state of mitochondrial cytochrome oxidase cannot be done in muscle becauseof Mb interference,60 whereas it is possible in the brain cortex.61

3.3

Which Variable Should be Considered?

Upon the premise that NIRS oxygenation signals reflect fractional O2 extraction (see below) in the investigated volumeof tissue, the following question can be asked: is there a correlation between NIRS-derived oxygenation signals and PO2 or
Hb saturation in venous blood draining from the exercisingmuscle(s)? The answer to this question may not be straightforward, and conflicting results have been reported in the past. The
issue is complicated by the problem of obtaining, particularly inhumans, adequate samples of venous blood coming directlyfrom the exercising muscle(s), without a “contamination”
from blood coming from other muscles and/or other tissues.
Wilson et al.62 found an excellent correlation between a
NIRS oxygenation index and O2 saturation determined in thevenous blood draining from a dog gracilis muscle preparation.
The data were subsequently confirmed in humans during forearm exercise.53 On the other hand, in two studies63,64 performed
during constant work rate exercise on a cycle ergometer, the following phenomena were observed (in normoxia): a transient
decrease in oxygenation following the transition to exercise,
which was paralleled by a decrease in femoral vein O2 saturation; these decreases were followed by a paradoxical muscle“reoxygenation,” which was not associated with an increased
O2 saturation in the femoral vein. The latter, as expected,
remained low for the remaining portion of exercise. In otherwords, following exercise onset an association between thetwo variables (decreased muscle oxygenation, decreased femoral vein O2 saturation) was observed, whereas dissociation
occurred during the remaining portion of the constant work
rate exercise. A similar muscle oxygenation pattern has been
observed in different muscles14,65,66 for the oxygenated-Hband -Mb ([oxy(Hb+Mb)]) signal (see below) while not for[deoxy(Hb+Mb)], which increased and then stayed constantly
elevated during the remaining portion of the exercise.

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Grassi et al.65 reasoned that the muscle reoxygenation suggested by the [oxy(Hb+Mb)] time-course could derive from anincreased blood flow to the skin, occurring for thermoregulatory
purposes, as also suggested by the results obtained by Maehara
et al.,67 Chuang et al.,68 and Davis et al.69 An increased skinblood flow for heat dispersion would increase the [oxy(Hb+Mb)] signal, whereas would not substantially affect [deoxy(Hb+Mb)], since the enhanced blood flow would not be associated with an increased gas exchange. This was substantiallyconfirmed in a study carried out by Koga et al.37 These authors
observed by continuous-wave NIRS (CW-NIRS) (see below andFerrari et al.24 for a definition), during whole body heating, amore pronounced increase of [oxy(Hb+Mb)] compared tothat of [deoxy(Hb+Mb)]. The apparent increased oxygenation
described by CW-NIRS was not observed when utilizing TDNIRS37 (see below). The confounding effects of an increased
skin blood flow on oxygenation variables [in particular [oxy(Hb+Mb)] determined by CW-NIRS] have been confirmed ina study carried out by Messere and Roatta.70
These authors observed that most of the warming-induced
increase of the sum between oxy- and deoxy-Hb and -Mb([total(Hb+Mb)]) resulted from an increase in [oxy(Hb+Mb)],
with a relatively small contribution by [deoxy(Hb+Mb)].
They also observed that the “contamination” of the muscle
NIRS signal by an increased skin blood flow was substantiallyreduced by using spatially resolved (SRS) NIRS (see below). No
increases in muscle O2 saturation (SO2m , see below) wereobserved, by utilizing SRS-NIRS, in the vastus lateralis of subjects performing dynamic knee-extension exercise at 20% of
MVC after thigh heating at 37°C and 42°C.71 The unchanged
SO2m was observed in the presence of a marked increase in cutaneous vascular conductance.54
Several studies have confirmed the presence of a good orvery good correlation between the [deoxy(Hb+Mb)] signal,or other tissue oxygenation variables determined by NIRS,
and venous O2 saturation, both in an animal model of isolated
muscle in situ14,72,73 and in exercising humans.20,30,74,75 In Wüst
et al.,72 the correlation was worse during the initial part of contraction period, which in that model is associated with a forceful“muscle pump” effect and a substantial blood squeezing resulting from the sudden onset of tetanic contractions.
The first NIRS instruments widely utilized on skeletal
muscle adopted, as a deoxygenation index, the differential
absorption of light between 760 and 800 (or 850)
nm.52,53,67,76–83 The approach was based on the absorption characteristics of NIR light by the chromophores of interest at thetwo wavelengths. Subsequent instruments allowed the obtainment of relative (with respect to an initial value arbitrarily set
equal to zero) or absolute [oxy(Hb+Mb)] and [deoxy(Hb+Mb)]
values. The sum of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] indicates the total Hb + Mb volume ([total(Hb+Mb)]) in the tissue
under consideration. Since total [Mb] cannot change acutely
during exercise, changes in [total(Hb+Mb)] would reflect a vasodilation or an increased capillary hematocrit in the tissue underconsideration. Absolute values of the above-mentioned variables can be obtained by the more technologically sophisticated(and more expensive) SRS, TD, or frequency-domain (FD)
instruments, whereas the less technologically sophisticated,less expensive, and more widely utilized CW instrumentsallow only relative values (for a more detailed discussion on
this topic, see below and Ferrari et al.18,21,24). This represents
a limitation, which can be at least mitigated by performing a
Journal of Biomedical Optics“physiological calibration” at the end of each test, by a transientischemia of the investigated limb, obtained by applying for a
few minutes a markedly suprasystolic pressure by a cuff,“upstream” of the region of investigation (see, e.g., Fig. 1 inPorcelli et al.84). This maneuver is not too uncomfortable forthe subject. The range of values of [deoxy(Hb+Mb)] obtainedin the muscle between rest and the condition of full extraction(i.e., when the [deoxy(Hb+Mb)] signal reaches a “plateau” after
4 to 5 min of ischemia) should be related to the range ofCða-vÞO2m values between rest and full extraction. By adopting
this physiological calibration, “semiquantitative” fractional
O2 extraction values can be obtained, also by CW-NIRS
instruments.
Some controversy has been raised85,86 about which one of thetwo variables ([oxy(Hb+Mb)] or [deoxy(Hb+Mb)]) should more
precisely reflect changes in skeletal muscle fractional O2 extraction. As discussed by Grassi,87 the time-course of [deoxy(Hb+Mb)] (while not that of [oxy(Hb+Mb)]) during a constantwork rate exercise65 qualitatively reflects, rather closely, thetime courses of variables related to fractional O2 extraction
across a wide spectrum of experimental models, such as
Cða-vÞO2 determined across a contracting isolated in situ
muscle preparation88 or an exercising limb,11,20 microvascular
PO2 (PO2mv ) determined by phosphorescence quenching in
rat spinotrapezius muscle,89 intracellular PO2 determined byphosphorescence quenching in isolated amphibian muscle
fiber.90 Similar kinetics between PO2mv and [deoxy(Hb+Mb)]
during electrically stimulated contractions in the rat gastrocnemius were observed.91 A study described a very similar pattern,also, for deoxy-Mb determined by 1 H-MRS17. In other words,
during constant work rate exercise (or isometric contractions ofconstant metabolic rate), a series of variables related to O2
extraction, spanning from Cða-vÞO2 across a whole limb to
Mb saturation, show a very similar time-course: unchanged
versus rest for a few seconds (suggesting an adequacy of_ 2m with respect to VO_ 2m —this information is relevant inQO_ 2 kinetics3,4,10), thenterms of the factors determining the VO_ 2m exceeds thea rapid exponential increase (the increase VO_ 2m ) to a new steady state, in which the ratio
increase in QO_ 2m reaches a new equilibrium, although at a higher_ 2m ∕QO
VO
value compared to that at rest. In other words, [deoxy(Hb+Mb)](while not [oxy(Hb+Mb)]) belongs to this family of variables
related to fractional O2 extraction. This indirectly but strongly
supports the role of [deoxy(Hb+Mb)] as an estimate of skeletal
muscle fractional O2 extraction. The concept is further strengthened by the issue of [oxy(Hb+Mb)] increases deriving from theenhanced blood flow to the skin for thermoregulatory purposes,
as discussed above.
Strictly speaking, an increased [deoxy(Hb+Mb)] can be considered an estimate of an increased fractional O2 extraction only
if [total(Hb+Mb)] is constant, which is not always the case in
exercising skeletal muscles. However, there is ample evidence14,18,65,86,92–94 suggesting that changes in [deoxy(Hb+Mb)] are less influenced by changes in [total(Hb+Mb)] compared to changes in [oxy(Hb+Mb)]. Adami et al.92 observed
a good correlation between the increase in [deoxy(Hb+Mb)]
and the decrease in femoral blood flow during passive head_ 2m .
up tilting, in the presence of a presumably unchanged VO
The data confirm the role of changes in [deoxy(Hb+Mb)] in
evaluating O2 extraction in the presence of acute and passively_ 2m . In the study, passive head-up tilt
induced changes in QO
induced also an increased venous blood volume, which

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complicated the interpretation of [deoxy(Hb+Mb)] as an index
of O2 extraction;92 venous blood pooling is, however, unlikely tooccur during exercise. In any case, the issue of the influence ofblood volume has been substantially overcome by a recent
study94 in which the authors calculated, on the basis of theassumption that during an arterial occlusion changes in [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] should occur with a 1∶1
ratio, a useful “blood volume correction factor,” which canbe applied to both [oxy(Hb+Mb)] and [deoxy(Hb+Mb)].
As mentioned above, SRS-, TD-, or FD-NIRS instrumentsallow the measurement of absolute values of [oxy(Hb+Mb)]
and [deoxy(Hb+Mb)].24 By utilizing these values, a tissue
oxygenation index (TOI) can be calculated, as the ratio [oxy(Hb+Mb)]/[total(Hb+Mb)].95 As an oxygenation variable, TOI
is appealing since it is expressed as a percentage, similarly toarterial O2 saturation (SO2a ). Since it includes [oxy(Hb+Mb)], however, also TOI may be more significantly influenced
by skin blood flow compared to [deoxy(Hb+Mb)].

3.4

Physiological Variables of Functional Evaluation

Which of the variables in the Fick equation [Eq. (1)] can be atleast estimated by NIRS? The answer is: all of them, although in_ m some specific (and rather_ 2m or Q
order to determine VO
unphysiological) or invasive protocols need to be adopted.
When these protocols are not utilized, the oxygenation indices_ 2mobtained by NIRS evaluate the dynamic balance between VO_ 2m . In other words, these indices allow the estimation of
and QO
skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m(see also below).
3.4.1

Skeletal muscle (or regional) blood flow:

De Blasi et al.96 determined forearm Q by NIRS, at rest and after
hand exercise, by inducing a 50-mmHg venous occlusion by acuff. They reasoned that if venous outflow from a muscle isimpeded by the cuff, whereas arterial inflow is not (or atleast is only partially impeded), the increase in [total(Hb_ The authors+Mb)] as a function of time should be related to Q.
_
observed indeed an excellent correlation between forearm Q,
determined by NIRS as described above, and the increase inforearm volume determined by plethysmography. A similar
approach was adopted by van Beekvelt et al.32 No comparison_ m was made. Limitations ofwith other methods to determine Q
the approach relate to the fact that pressure by the cuff inevitably
limits, although only in part, also arterial inflow, as recently_ measurements could be carconfirmed.97 Moreover, regional Q
ried out only during the recovery from exercise, and it is wellknown that the variable may rapidly change in the recovery
phase.6_ m in the exercising calf muscles has been determined inQhumans by utilizing NIRS in association with the intravenous
infusion of the NIR tracer indocyanine green (ICG).98 ICG isa fluorescent dye approved by the U.S. Food and Drug
Administration and European Medicines Agency for humanuse. Arterial blood was withdrawn by a pump and [ICG] wasdetected by photodensitometry, whereas NIRS optodes positioned over the muscle detected ICG transit in the microcircu_ m measurements were comparedlation at several wavelengths. Q
with those obtained by established methods. The main drawbackof this elegant approach resides in its invasiveness, related to theneed of obtaining arterial blood samples. The method has been
_ m in respiratory99 and skeletal muscles100.
utilized to determine Q
Journal of Biomedical Optics

Vogiatzis et al.30 utilized it to evaluate the intramuscular match_ 2m .
_ 2m and QO
ing between VO
3.4.2

Skeletal muscle O 2 consumption

Hamaoka et al.101 determined by NIRS the initial rate of Hb andMb deoxygenation in finger flexors of humans during ischemia
performed at rest and immediately after submaximal exercise ofdifferent intensities, and considered this variable a reflection of_ 2m . These authors observed a significant correlation betweenVOthis rate and the concentrations of intramuscular metabolites(PCr and adenosine diphosphate), determined by 31 P-MRS, considered to be regulators of oxidative phosphorylation. They concluded that NIRS can quantify the rate of oxidative metabolism
at rest and after exercise. The validity of the approach was subsequently confirmed.102 The limitations are represented by thefact that the NIRS measurements need to be carried out in ischemic conditions and that the measurements are possible only in_ 2 rapthe recovery phase following exercise, during which VO
6idly decreases.
While ischemic contractions do not represent a physiologicalcondition, a “less unphysiological” experimental model would
be one in which blood flow is held constant. Also in this case,according to the rearranged Fick equation [Eq. (2)], and after
considering [deoxy(Hb+Mb)] a proxy of Cða-vÞO2m , theincrease in [deoxy(Hb+Mb)] should represent an estimate of_ 2m . This has indeed been experimentally confirmed in theVOstudy by Wüst et al.,72 which carried out in an isolated dog gas_ m was held controcnemius in situ preparation. In the study, Q_ 2m was calculated by the Fick equation
stant by a pump, VO
across the muscle and NIRS optodes were directly applied on
the surface of the exposed muscle. During metabolic transitions,the kinetics of adjustment of [deoxy(Hb+Mb)] was very similar_ 2m .
to the kinetics of adjustment of VO
Brief sequential ischemic periods carried out during therecovery from exercise/contractions have been utilized inorder to determine, by NIRS, the kinetics of recovery of_ 2m following exercise.94,103–106 Also in this case, the rateVOof increase in [deoxy(Hb+Mb)] (and/or the rate of decreasein [oxy(Hb+Mb)]) during each of the brief ischemic periods_ 2m . The kinetics of recovery
is considered an estimate of VO_ 2m , as determined by the abovementioned protocol, hasof VObeen shown to be closely correlated with two well-establishedvariables of functional evaluation of skeletal muscle oxidative
metabolism (frequently and incorrectly termed “oxidative
capacity” by some of these authors): (1) the kinetics of recoveryof [PCr], as determined by 31 P-MRS following exercise104 and(2) maximal adenosine diphosphate-stimulated mitochondrial
respiration, as determined by high-resolution respirometry inisolated and permeabilized skeletal muscle fibers.106
3.4.3

Skeletal muscle fractional O 2 extraction

As discussed above, in “normal” experimental conditions, oxygenation variables obtained by NIRS allow the evaluation of the_ 2m , in other words of_ 2m and QO
dynamic balance between VO
skeletal muscle fractional O2 extraction, a proxy of Cða-vÞO2m .
NIRS oxygenation variables can represent only a proxy of
Cða-vÞO2m since they are usually expressed in relative terms,
and the proportional contributions of arterial and venous
blood to the overall signal are unknown. In the next paragraphs,
we will provide examples of conditions in which analysis of091313-7

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skeletal muscle fractional O2 extraction appears relevant and ofphysiological interest.
The capacity for prolonged exercise depends upon the capacity to supply the exercising muscles with O2 in order to satisfy
the metabolic requirements. During exercise, however, fractional O2 extraction increases. In other words, compared torest, the muscles (as other organs) utilize a greater percentageof the O2 they receive by the cardiovascular system. We will
discuss, later in this section, how this increase in fractional
O2 extraction could impair peripheral O2 diffusion and oxidative
metabolism through the resulting decrease in microvascular PO2(PO2mv ). Textbook physiology says that at the systemic level, in
humans at rest, fractional O2 extraction is ∼25%, whereas during maximal exercise, the variable increases to ∼75%. In steady_ andstate conditions during exercise, the relationship between Q_ 2p is linear, with a slope of 5 (1 L min−1 increase in VO_ 2p isVO_ Considering that
accompanied by a 5 L min−1 increase in Q).
∼1∕5 of the volume of arterial blood is represented by O2(CaO2 ∼ 200 mL L−1 of blood), the linear relationship between_ 2 and VO_ 2p has a slope of 1. As a consequence of the linear
QO_ (or QO
_ 2 ) versus VO_ 2p relationship, systemic Cða-vÞO2 would
Q
increase hyperbolically as a function of work rate. This has
indeed been observed during ramp incremental cycling
exercise.107_ m O2 and VO_ 2m is somehow differThe relationship between Q
ent when determined at the microvascular level, which is thedomain investigated by NIRS. During an incremental or ramp
exercise, the NIRS-derived [deoxy(Hb+Mb)] follows a sigmoid
pattern,62,84,107–113 with a shallow increase at low work rates (sug_ 2m , pre_ 2m and QO
gesting a tighter matching between muscle VO
sumably associated with the recruitment of more oxidative
fibers), a linear intermediate portion (suggesting greater O2_ 2m, presumably_ 2m ∕QO
extraction and a progressively higher VO
associated with the recruitment of less oxidative fibers), followed_ 2m;max .
by a plateau at work rates approaching ∼80% of VO
At submaximal work rates, before the “plateau” is reached,
the observation of higher fractional O2 extraction for the same_ 2m (or work rate), in other words, a left-shifted [deoxy(HbVO_ 2m .
+Mb)] versus work rate curve would suggest an impaired QO
On the other hand, a lower fractional O2 extraction (right-shifted[deoxy(Hb+Mb)] versus work rate) would suggest an excess_ 2m with respect to VO_ 2m . This information may have proQO
found physiological and pathophysiological significance (seebelow). The sigmoid shape of the [deoxy(Hb+Mb)] versus
work rate curve has been shown to vary in muscles characterized
by different fiber type compositions49 and has been confirmed(with minor differences) also during supine exercise114 as well as
during incremental exercise in acute hypoxia.115 A right-shifted[deoxy(Hb+Mb)] versus work rate curve was described intrained cyclists.116
The [deoxy(Hb+Mb)] kinetics during constant work rate
exercise has been utilized to evaluate the effects on the rateof adjustment of oxidative metabolism by a “prior” or “warmup” or “increased baseline” exercise,72,117–119 or to investigate
the effects on microvascular fractional O2 extraction by interventions such as dietary nitrate supplementation, aimed atincreasing microvascular nitric oxide (NO) availability.120 For
example, the increased muscle oxygenation observed by
Gurd et al.118 following a prior heavy intensity exercise suggests
an enhanced O2 availability, which should have contributed to_ 2 kinetics. In other studies,
the observed faster pulmonary VO
the results were not conclusive. Bailey et al.,117 for example,
Journal of Biomedical Optics

observed after oral nitrate supplementation, a decreased muscle
O2 extraction, which, however, could not be simply attributed
to enhanced O2 delivery since it was associated with a decreased_ 2.
VO
Skeletal muscle fractional O2 extraction determined by NIRS
can also give valuable physiological and pathophysiologicalinsights into the integrated responses to exercise (and to otherconditions) involving different muscle groups or skeletal
muscles and other organs, as well as into the physiological_ m regulation.
mechanisms of Q
_ (by thermodilution) and muscle oxyFor example, arm Q
genation (by NIRS) were measured at the transition from_arm to arm + leg exercise.66 During maximal exercise, arm Q_ 2m;max and muscle
decreased at the transition, to a degree that VO
oxygenation were compromised. Thus, NIRS can give valuable_ m and VO_ 2m adjustments toinsights into the coordination of Q
exercise between different muscle groups, in conditions of_ 2m (see also below).
increased O2 demands and/or limited QO
Turner et al.121 observed [deoxy(Hb+Mb)] increases both inlimb locomotor muscles and in respiratory muscles after interventions, such as inspiratory muscle loading performed during_ 2p;max , or after increasing work
cycling exercise at 80% of VO_ 2p;max . The results suggest a limrate from 80% to 100% of VO_ 2m , both in locomotor and in respiratory muscles, whichited QO
may impair exercise tolerance during maximal exercise or in
conditions of increased respiratory muscles work.
By applying lower body negative pressure, Soller et al.122
induced a progressive decrease of central blood volume and adecreased stroke volume, which were paralleled by a decreased
muscle oxygenation from vasoconstriction. According to theseauthors, skeletal muscle deoxygenation determined by NIRS
could be utilized as an early indicator of central hypovolemia
and impending cardiovascular collapse.
NIRS has also been utilized to elucidate basic physiological_ m regulation, such as the metabolic modulation
mechanisms of Q
of sympathetic vasoconstriction (functional sympatholysis) by
exercise123 or by tissue hypoxia.124 By simultaneously measuring sympathetic nerve activity by microelectrodes and forearm
oxygenation by NIRS, Hansen et al.123 provided evidence infavor of functional sympatholysis. Reflex sympathetic activation, which consistently decreased oxygenation in resting forearm muscle, had no effect on oxygenation when the muscles
were exercised.
The [deoxy(Hb+Mb)] plateau during an incremental exercise
can be considered an estimate of the maximal capacity of O2
extraction during exercise and has been utilized in several
studies84,109–113,125–127 as a variable of functional evaluation ofskeletal muscle oxidative metabolism.
Venous blood draining from exercising muscles still contains
small but significant amounts of O2 ; in other words, fractional
O2 extraction is not 100%.128–130 Which are the normal values offractional O2 extraction across skeletal muscle during maximal
exercise? According to Knight et al.,128 during cycling, the value
can go up to ∼90%, as determined by invasive measurements
carried out across an exercising limb. A fractional O2 extraction
value of ∼85%, also in this case determined invasively across an
exercising limb, was observed during maximal knee-extensor
exercise,130 which is an exercise paradigm (involvement of a relatively low small muscle mass) in which cardiovascular constraints to O2 delivery are not present or are significantly
reduced, and skeletal muscle blood flow can be three times
higher than during conventional cycle ergometer exercise.

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These values are very similar to those obtained in normal subjects by NIRS for [deoxy(Hb+Mb)] at peak exercise, expressed
as a percentage of the maximal [deoxy(Hb+Mb)] value observed
during a transient limb ischemia.84_ 2p;max is traditionally conAlthough, in normal subjects, VO_ max )
sidered to be mainly limited by maximal cardiac output (Q_ 2 max ),131 the role ofand maximal cardiovascular O2 delivery (QO
peripheral factors cannot be overlooked.132,133 If one considers
the Fick equation [see Eq. (1)] applied to conditions of maximal
exercise, it is obvious that the main difference between subjects_ 2p;max and subjects with low VO_ 2p;max resides inwith high VO_
QO2 max , and not in Cða-vÞO2 max . However, the concept that_ 2 max represents cardiovascular limitations and Cða-vÞO2 max
QO
or maximal fractional O2 extraction represent peripheral factors
is an oversimplification.134 In an in-series O2 transport system,
every step must play a role in affecting the overall outcome, and_ 2 , bythe different functions are strictly interconnected.133 QO
setting microvascular PO2 (PO2mv ), determines peripheral O2
diffusion and therefore fractional O2 extraction. In the presence_ 2 , as it occurs in athletes or after training,of an elevated QO
peripheral O2 diffusing capacity (DO2m ) must increase inorder to allow a large fractional O2 extraction.
Although the issue is debated,135 in skeletal muscle, an O2_ 2p;max and
extraction limit is present, which contributes to VO_ 2m;max limitation; moreover, a limitation in peripheral O2 difVO
fusion appears to be the major factor responsible for the limited
extraction.133 Exercise training may increase muscle fractional
O2 extraction by elevating muscle(s) DO2m to a greater extent_ 2m;max .35,42 Kalliokowski et al.136 observed by positron
than QO
emission tomography that fractional O2 extraction during exercise is higher in endurance trained athletes compared tountrained controls. According to Whipp and Ward,137 systemic_ 2p;max . In otherCða-vÞO2 max increases hyperbolically with VO
words, also in healthy subjects, fractional O2 extraction is rel_ 2p;max . This concept applies even more
evant in determining VO
strongly to patients or to conditions in which oxidative metabolism is impaired (see below).
The “scenario” depicted above can be looked at also fromanother perspective: an increased O2 extraction may impair
peripheral O2 diffusion. This could occur also during submaximal exercise. A fall in oxygenation (corresponding to an_ 2m ) suggests a less pronounced or inad_ 2m ∕QO
increased VO_ 2m .
equate vascular response in relation to an increased VO
This would determine a decreased PO2mv and mean capillary
PO2 , a decreased driving pressure for peripheral O2 diffusionfrom capillaries to mitochondria,42,43,138 and a functional impairment of skeletal muscle oxidative metabolism.
The phenomenon could also occur transiently in the earlyphase of a constant work rate exercise. This is suggested by
the transient “overshoot” of skeletal muscle fractional O2 extraction (usually determined by analyzing the [deoxy(Hb+Mb)]
time-course), which has been described in contracting in situ
dog muscle14 as well as during constant work rate exercise indifferent populations (see also below), such as patients withchronic heart failure (CHF),95,139 patients with metabolic
myopathies,140 type 2 diabetes,141 chronic obstructive pulmonary disease,142 healthy subjects undergoing bed rest
deconditioning84 or bed rest associated with hypoxia,127 healthy
subjects during arm exercise.81 In the latter study, the overshootin skeletal muscle fractional O2 extraction, determined by NIRS,
was closely associated with an overshoot in Cða-vÞO2 , determined invasively across the exercising muscles. Goodwin
Journal of Biomedical Optics_ 2m kinetics in isolated in situ
et al.14 experimentally slowed QO
dog muscle and observed an enhanced [deoxy(Hb+Mb)] over_ 2m kinetics. In human subjects acutely
shoot and a slowed VO
exposed: to normobaric hypoxia, Bowen et al.33 observed aslower VO2p kinetics and a more pronounced [deoxy(Hb+Mb)]
transitory increase, although the magnitude of the overshoot was
unaffected. The O2 extraction overshoot, occurring early during
a constant work rate exercise, is considered to be caused by a
_ 2m and VO_ 2m .42,43,138,143,144 The
transient mismatch between QO
mismatch could derive from a suboptimal NO signaling within
the muscle, which could “uncouple,” both temporally and spa_ 2m increase from thetially, the heterogeneous microvascular QO_
presumably heterogeneous VO2m increase.43,138
The transiently increased fractional O2 extraction is associated with a transiently decreased PO2mv .42,43,138,144 This phenomenon has been described in animal models, in which
PO2mv was determined by phosphorescence quenching.35,145
As mentioned above, the decreased PO2mv would reduce thedriving pressure for peripheral O2 diffusion and impair oxidative
metabolism. The impairment would be functionally significant
also because it would be temporally located in the critical earlyphase following an increase in metabolic intensity.3,4,6,10
A partially different approach to the [deoxy(Hb+Mb)] overshoot has been taken by Murias et al.146 These authors utilized_ 2p ratio, in which botha “normalized” ½deoxyðHb þ MbÞ∕VO
variables are expressed as a percentage of the response. In_ 2p signal is left-shifted byorder to calculate the ratio, the VO
20 s, which is assumed to represent, on average, the delay_ 2m increase (the variable of interest in the combetween the VO_ 2p increase,3,4 which
parison with [deoxy(Hb+Mb)]) and the VO
is the variable usually determined. Another assumption related_ 2p kinetics equals VO_ 2m kinetics. Thisto this approach is that VO
is not exactly true (see the discussion in Koga et al.42): the twovariables have similar kinetics11 but with a substantial intersub_ 2p increases
ject variability. Moreover, [deoxy(Hb+Mb)] and VO
are, in relative terms, substantially different, and therefore, thenormalization (percentages of responses) likely introduces a distortion in the calculation of the ratio.
In another approach, Ferreira et al.143 assumed (as Murias_ 2p kinetics equals VO_ 2m kinetics, andet al.146 did) that VO
that [deoxy(Hb+Mb)], as an estimate of O2 extraction, quantitatively reflects Cða-vÞO2 ; the authors then calculated microvascular blood flow adjustment by solving the Fick equation. The
main limitations of this approach, in our opinion, reside in the_ 2m kinetics equals to VO_ 2passumptions: the first one (VOkinetics) has been discussed above, but also the second one([deoxy(Hb+Mb)] as a quantitative measurement of Cða-vÞO2 )is not strictly correct: even when convincing “physiological calibration” methods are utilized, [deoxy(Hb+Mb)] allows at best
only a semiquantitative estimation of Cða-vÞO2 , and therefore,
its use in replacing Cða-vÞO2 in mathematical equations seems
questionable.147 Moreover, the approach by Ferreira et al.143
assumes that the contribution by Mb to the NIRS signals iszero; as discussed above, this is likely not the case.
_ 2p
During the recovery following exercise, the decrease in VO_ 2m follows an exponential time course, even in theand VO
presence of a sudden decrease in mechanical power output.6_ 2m ,” with respect to the
In these conditions, the “excess VO
needs of myosin ATPase, derives essentially from the resynthesis of PCr, which occurs by oxidative phosphorylation. Upon
the assumption of equilibrium of the CK reaction, the recovery
rate of [PCr] following exercise is therefore a function of

091313-9September 2016 • Vol. 21(9)Grassi and Quaresima: Near-infrared spectroscopy and skeletal muscle oxidative function. . .

mitochondrial ATP production. The rate of recovery of [PCr]following exercise has often been utilized as a tool of evaluationof skeletal muscle oxidative metabolism.15,104 Another variablefrequently utilized as a functional evaluation tool is the NIRSdetermined reoxygenation kinetics during the recovery. This
kinetics is a function of the interplay between the kinetics of_ 2m and the kinetics of VO_ 2m , and cannot discriminate
QO
between the two. However, a good agreement was foundbetween the rate of recovery of [PCr] (as determined by
31 P-MRS) and the reoxygenation kinetics (as determined by
NIRS) following moderate-intensity exercise that is the absenceof significant drops in pH.80 These authors concluded that thereoxygenation kinetics during the recovery from a moderateintensity exercise could represent a functional evaluation toolof oxidative performance.
3.4.4

Other variables and purposes

versus young activity-matched controls, in association with_ 2m and an unchanged microvascular frac_ m and QO
higher Q
tional O2 extraction. These results point to a higher energy
cost of contractions as the main factor responsible for the
reduced exercise capacity in the elderly. At least in part, different
results were described by Ferri et al.,125 who observed in 78-year
old subjects, higher skeletal muscle (vastus lateralis) fractional
O2 extraction, at the same absolute work rate, suggesting an_ 2m . This phenomenon was observed both durimpairment of QO
ing cycle ergometer exercise (in which the limitations to oxidative metabolism are mainly related to cardiovascular O2
delivery) and during one-leg knee-extension exercise,125 inwhich cardiovascular limitations are substantially reduced.130_ 2p kinetics is slower in the elderly. Murias et al.159
VOobserved that this slower kinetics was associated with a larger_ 2p (both variables wereovershoot in the ½deoxyðHb þ MbÞ∕VO
expressed as a percentage of their total response, see also above)
ratio, suggesting, according to the authors, an impaired adjust_ 2m . This would be in agreement with observations ofment of QO
a blunted vasodilatory dynamics in arterioles of old rats.160_ 2p kinetics and reduced theExercise training speeded VO_½deoxyðHb þ MbÞ∕VO2p overshoot,159 suggesting an improved_ 2m and VO_ 2m . In another study, no
matching between QO_ 2p overshoot was observed in chroni½deoxyðHb þ MbÞ∕VO
cally trained elderly subjects.161 It should be considered, however, that in these studies,159,161 the overshoot in [deoxy(Hb_ 2 ; in other words, no “real”+Mb)] was only relative to VO[deoxy(Hb+Mb)] overshoot was present.

Several studies have been conducted with the aim to identify, by
NIRS, variables of functional evaluation of oxidative metabolism (thresholds), such as the ventilatory threshold,148,149 the“EMG threshold,”150 the onset of blood lactate accumulation
during incremental exercise,151 or the “maximal lactate steadystate.”152 Most of these studies observed a nice correlation
between NIRS-obtained indices and more traditional variables
utilized to identify the various thresholds.148,149,151,152
Broxterman et al.153 utilized NIRS, together with EMG and
the analysis of the curvature constant of the power versus time toexhaustion relationship, in order to evaluate the role of central
and peripheral fatigue during exercise with small muscle mass
(handgrip) in conditions of blood flow occlusion. Central (brain)
factors were identified as responsible for fatigue during submaximal elbow flexors exercise in severe hypoxia;154 in the study,
the authors utilized muscle and brain NIRS, evoked and EMG
responses to electrical muscle stimulation, transcranial magnetic
stimulation, and motor-evoked potentials.
Soller et al.122 developed a mathematical model, which correlated NIRS spectra with muscle pH (pHm ) values obtained
invasively by sensors placed in the muscle. They could then predict pHm values on the basis of NIRS spectra, and determined,
during incremental exercise, an “Hþ threshold,” which correlated with classic “metabolic thresholds”.
Towse et al.155 utilized NIRS blood volume and muscle oxygenation changes to confirm the blood-oxygenation-leveldependent (BOLD) mechanism underlying the changes ofmagnetic resonance imaging signal intensity observed following
brief, single contractions.
Several NIRS studies have been performed in electricallystimulated contractions. For instance, McNeil et al.156 observed
by NIRS a greater metabolic demand, associated with greater
fatigue during electrically stimulated (versus voluntary), contractions carried out at the same torque production. On the contrary, the local O2 demand of the biceps brachii was foundsimilar between electrical muscle stimulation and maximal voluntary isometric contractions.157

Higher skeletal muscle fractional O2 extraction for the same
work rate, a reduced peak capacity of O2 extraction during
an incremental exercise, and an overshoot of [deoxy(Hb+Mb)]
during constant work rate exercise were observed in healthy
young subjects undergoing 35 days of bed rest.84,111 These findings were observed during both cycle ergometer exercise84 andone-leg knee extension exercise.111 These results include skeletal muscle oxidative metabolism among the “victims” of profound inactivity. Since bed rest studies have been considered a“proxy” of microgravity (see the discussion in Porcelli et al.,84
Salvadego et al.111), the same concept applies to microgravity.
Superposition of normobaric hypoxia (equivalent to an altitude
of about 4000 m) upon 10 days of bed rest attenuated, during
one-leg knee extension exercise (but not during cycle ergometer
exercise), the impairment of the peak capacity of fractional O2
extraction and the magnitude of the overshoot, which wereobserved following bed rest alone.127 An enhanced perfusiveand diffusive O2 delivery deriving from the hypoxia-induced
increased [Hb] may explain, at least in part, this unexpected
finding.
Training programs carried out during 21-day forearm immobilization by a plaster cast were found effective in preventing thedecline in muscle oxidative function, endurance, and strength.162

4Aging, Immobilization, Hypoxia

4.3

4.1

Aging

By combining 31 P-MRS, Doppler ultrasound imaging, andNIRS Layec et al.158 observed a higher ATP cost of contractions
during plantar flexion exercise in elderly subjects (73-year old)
Journal of Biomedical Optics

4.2

Immobilization/Microgravity andCountermeasures

Hypoxia

During acute moderate normobaric hypoxia (inspired O2 partialpressure [PO2I ] 118 mmHg, equivalent to an altitude of∼2500 m), muscle deoxygenation during an incremental
cycle exercise is not significantly different, for the same absolutework rate, compared to normoxic conditions.163 The data

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_ m in hypoxia compared to normoxia and, thus,
suggest higher Q
the existence of control mechanisms, which effectively match_ 2m to VO_ 2 . This effective matching is presumably lost at
QO
higher altitudes. Billaut et al.164 collected muscle and brain oxygenation data to gain insights into the central and peripheral factors affecting muscle fatigue in conditions of limited O2delivery. They described a trend toward lower vastus lateralis
muscle oxygenation in subjects performing repeated sprints
and acutely exposed to a slightly more pronounced (comparedto that of the previously mentioned study) moderate hypoxia(FO2I ¼ 0.138, PO2I 98.4 mmHg). Subudhi et al.165 described
greater fractional O2 extraction values for the same absolutework rate in subjects acutely exposed to hypobaric hypoxia(PO2I 86 mmHg) corresponding to an altitude of ∼4300 m.
Interestingly, no differences versus acute hypoxia were
described, in the same subjects, in chronic hypoxia (5-day acclimatization at 1830 m and 1-day at 4300 m). In all the mentioned
studies,163–165 marked differences between normoxia and acuteor chronic hypoxia were described for prefrontal cortex
oxygenation.
Unchanged muscle oxygenation during exercise wasdescribed after versus before a 72-day acclimatization to chronic
hypobaric hypoxia (Mt. Everest expedition);166 the unchanged
muscle deoxygenation was associated with an increased
hypoxic tolerance and with enhanced acute hypoxic ventilatory
response and cerebral oxygenation. In another Mt. Everest
expedition, a slower muscle (thenar eminence) reoxygenation
kinetics was observed during vascular occlusion tests carriedout at 4900 and 5600 m.167
A more pronounced muscle deoxygenation during constantwork rate exercise was described during acute hypoxia,67
obtained either by having the subjects breath air with a FO2I
of 0.15 or by carbon monoxide loading aimed at increasing carboxyhemoglobin saturation. Interestingly, the accelerated deoxygenation was described only during exercise above the “lactate
threshold,” that is, at an exercise intensity at which O2 supply tothe exercising muscles becomes critical.2,3,4,10
A greater deoxygenation of accessory respiratory muscles
during voluntary isocapnic hyperpnea was observed in hypoxia(FO2I ¼ 0.10 − 0.11) versus normoxia,168 in association with ahigher muscle activation determined by EMG.

5

Patients Populations

In this section, we will present an overview of some representative studies in which the variables of functional evaluation discussed in the previous section have been determined in adultpatients’ populations. The aims were to evaluate the evolutionof impairments as a function of time, to identify correlations
with clinical symptoms, to evaluate the effects of therapeuticor rehabilitative interventions, and to gain pathophysiologicaland diagnostic insights.
A systematic review on muscle NIRS measurements in theclinical care of term and preterm neonates has been recently
published.169

5.1

Chronic Heart Failure

Several studies have been conducted by utilizing NIRS inpatients with CHF. The complex interplay between central
and peripheral factors in determining the pathophysiology ofthe disease and the impaired exercise tolerance in CHF patientshas been discussed in detail in the review by Poole et al.138
A subsequent review by the same group144 discussed, from
Journal of Biomedical Optics

the same perspective, the effects of training in this patient
population.
Wilson et al.62 observed higher fractional O2 extraction, forthe same absolute work rate, in CHF patients versus agematched healthy controls; the results suggest an impaired_ 2m in the patients, presumably a consequence of the impaired
QO
cardiac function. A similar pattern was observed by anotherstudy in CHF patients.126 Interestingly, in this study, the_ 2m response was not affected by 3 months ofimpaired QO
light-to-moderate exercise training. The training intervention,on the other hand, increased the maximal capacity of skeletal
muscle fractional O2 extraction during exercise, allowing thepatients to reach values not different from those of controls.
A very similar pattern to that described in CHF patients126
before training was observed in patients evaluated after a successful heart transplantation,109 carried out in the treatment ofsevere CHF. The data suggest that the surgical intervention,although improving cardiac function, did not significantly_ 2m impairment at submaximal exercise andimprove the QO
the reduced capacity of maximal fractional O2 extraction byskeletal muscles. Also in patients undergoing heart transplantation, a complex interaction between central and peripheral functions limits exercise tolerance.109
Sperandio et al.139 described by NIRS in CHF patients, during
constant work rate submaximal exercise, a transient overshoot in
fractional O2 extraction (increased [deoxy(Hb+Mb)]), presumably leading to a decreased PO2mv and to an impaired peripheral
O2 diffusion during the critical early phase of the metabolic transition. This overshoot could derive from a suboptimal intramuscular NO signaling.138 The [deoxy(Hb+Mb)] overshoot isqualitatively equivalent and has the same pathophysiologicalmeaning with respect to the transient PO2mv undershoot describedin the spinotrapezius muscle of CHF rats.170 In addition,
Sperandio et al.139 observed that in CHF patients an increased
NO bioavailability, obtained by the administration of sildenafil,
eliminated the [deoxy(Hb+Mb)] overshoot, determined a faster_ 2p kinetics, and improved exercise tolerance. Bowen et al.95
VOobserved that the deoxygenation overshoot in CHF patientswas attenuated by a prior exercise (aimed at enhancing_ 2m ), which determined also a faster VO_ 2p kinetics, confirming
QO
the link between the overshoot and an impaired oxidative metabo_ 2p kinetics, thelism. On the basis of the deoxygenation and VO
authors identified two subgroups of patients: the most severely
limited ones were those with an impaired skeletal muscle oxidative metabolism.95
Belardinelli et al.82 observed in CHF patients a slower reoxygenation kinetics following exercise, in association with a_ 2p kinetics. In another study, Hanada et al.171
slower VO
observed that the slower reoxygenation kinetics in CHF patientswas associated with a markedly slower kinetics of recovery of[PCr], as determined by 31 P-MRS. As mentioned above, aslower reoxygenation kinetics during the recovery after exercise
is considered an index of impaired skeletal muscle oxidative
performance.80 At least in part, different results were obtainedby Kemp et al.:172 according to these authors, in CHF patientsthe slowing of muscle reoxygenation kinetics was more pronounced than the slowing of [PCr] recovery; the data suggest_ 2m limitation.
a QO_ 2m (determined by NIRS by
A slower recovery kinetics of VO
utilizing the method by Ryan et al.94 mentioned above) following wrist-flexor exercise was observed in systolic CHF
patients;173 interestingly, whereas in control subjects, a 4-

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week period of exercise training determined a faster recovery
kinetics, this did not occur in the CHF patients. The authors concluded that the impairment of nonlocomotor muscles oxidative
metabolism in the CHF patients was not reversible with a shortperiod of exercise training. Moreover, the impairment was not
simply due to deconditioning, but suggests a skeletal muscle
dysfunction intrinsically associated with the disease, as also proposed in the reviews by Poole et al.138 and Hirai et al.144
Mancini et al.76 observed deoxygenation of accessory
muscles of respiration (serratus anterior) in CHF patients(but not in controls) during submaximal and maximal cycle
exercise; the observed deoxygenation may contribute to theexertional dyspnea typically described in these patients. It is
well known that more severely affected CHF patients typicallyhave a hyperventilatory response to exercise, which holds
clinical and prognostic implications.174 The hyperventilatory
response, the decreased respiratory muscle strength, and the_ 2m requirements
increased work of breathing would increase QO
of respiratory muscles, and could induce a “competition” for the_ 2 between respiratory and locomotor
limited available QO
muscles, contributing to the decreased exercise tolerance.175
In support of this hypothesis, Borghi-Silva et al.176 observedthat respiratory muscles unloading, obtained in CHF patientsby proportional assisted ventilation, increased exercise tolerance
in association with an enhanced leg muscle oxygenation andblood volume determined by NIRS. These studies demonstrate
how NIRS can give valuable pathophysiological insights into
the interplay between different muscles in conditions of limited_ 2.
available QO
A functional evaluation of skeletal muscle oxidative metabolism would be even more important in CHF patients with preserved ejection fraction, in whom peripheral impairments appear
more significant than those usually observed in the more “traditional” CHF patients with reduced ejection fraction.144,177 In thisrespect, very few data are available yet.

5.2

Peripheral Arterial Disease and Diabetes

During incremental exercise, PAD patients show near-maximal
deoxygenation (with respect to the ischemic calibration) beginning from very low work rates.79 In the same study, the reoxygenation kinetics following exercise was markedly slower in
PAD patients; moreover, the half-time of this recovery was significantly correlated with the “ankle-arm index” (ratio ofsystolic blood pressures at the ankle and at the arm),79 a clinicalvariable evaluating the severity of the disease. A markedly
slower reoxygenation kinetics in PAD patients was confirmed
in subsequent studies.83,141
In PAD patients, the involvement of the two legs is often
markedly asymmetrical. Interestingly, McCully et al.79 observedthat the reoxygenation kinetics was markedly slower in the“bad” leg versus the “good” leg. According to McCully
et al.,83 analysis of reoxygenation kinetics by NIRS could be
utilized as a screening tool for the presence of PAD.
In patients with PAD, NIRS has also been utilized to evaluate
the effects of various therapeutic interventions. Whereas Beckitt
et al.178 described only modest improvements of muscle oxygenation during exercise in PAD patients treated with angioplasty, substantial positive effects were observed after theadministration of sildenafil179 or after an oral supplementation(beetroot juice) of nitrates.180 Both these approaches aimed,
although by exploiting different pathways, at increasing NO bioavailability in the microvasculature, thereby taking advantage of
Journal of Biomedical Optics

the vasodilatory effect of NO and also at improving the intra_ 2m .
_ 2m and QO
muscular matching between VO
What is the pathophysiological meaning of the slower reoxygenation kinetics in PAD patients? As a general rule, thiskinetics is a function of the interplay between the kinetics of_ 2m and the kinetics of VO_ 2m during the recovery following
QO
exercise. In PAD patients, the first variable is obviously
impaired, but the second one could be impaired as well. Thishas been demonstrated by Bauer et al.,141 who observed in
PVD patients a slower recovery of reoxygenation also in thepatients with reasonably normal calf blood flow responses, as
evaluated by plethysmography.
The clinical relevance of NIRS applications in PAD has beenvery recently reviewed by Boezeman et al.,181 who considered
183 manuscripts. These authors found sufficient evidence to use
NIRS in the clinical setting for assessment of chronic compartment syndrome of lower extremities, and as a surveillance tool
for the detection of “free flap failure”. On the other hand, theclinical relevance of routine use of NIRS in other vascular diseases was found to be less clear.
_ 2p , type 1 diabetic
For the same absolute work rate and VO
patients show (versus controls) higher [deoxy(Hb+Mb)], sug_ m.182_ 2m , as also suggested by the lower Q
gesting an impaired QO
An overshoot of [deoxy(Hb+Mb)] during constant work rate
exercise has been described in patients with type 2 diabetes.183_ 2p kinetics andThis observation was associated with slower VO_ 2m adjustment, conseappears compatible with an impaired QO
quence of the microvascular impairment. The [deoxy(Hb+Mb)]
overshoot described in diabetic patients is qualitatively equivalent and has the same pathophysiological meaning of the transient PO2mv undershoot described in the exercising muscles ofdiabetic rats.89
In a review, Pedersen et al.184 discussed the main methods toevaluate mitochondrial dysfunction in patients with PAD andtype 2 diabetes. According to these authors, NIRS works better
than 31 P-MRS in determining the severity of the impairment.
Manfredini et al.185 utilized NIRS in order to find objective
correlates between the onset of symptoms of claudication andthe limited exercise tolerance in PAD patients, with and without
diabetes. In order to do so, they determined the area under the[oxy(Hb+Mb)] or [deoxy(Hb+Mb)] curves (with respect to thebaseline) during an incremental exercise. These areas evaluate(although in arbitrary units) the overall burden represented by_ 2m . The areas_ 2m and QO
the dynamic unbalance between VO
were greater in the PAD patients with diabetes and were correlated with the onset of symptoms. In the PAD patients with diabetes, however, ischemic symptoms were delayed and occurred
at a level of deoxygenation which was significantly greater thanin the patients with PAD alone, possibly as a consequence of ablunted pain perception and/or of a subjective higher tolerance
for muscle discomfort associated with diabetes.

5.3

Chronic Obstructive Pulmonary Disease

By substituting nitrogen with helium in inspired air, gas density
is reduced to ∼1∕3 of that of normal ambient air. After relieving
the work of breathing by having COPD patients breathe a normoxic helium-O2 mixture, Chiappa et al.186 observed less leg_ 2p kinetics, and an increased
deoxygenation by NIRS, faster VO
exercise tolerance. The authors hypothesize that by decreasing_ 2 between respiratory andthe competition for the available QO
locomotor muscles, helium-O2 breathing could increase leg_ 2m , thereby enhancing skeletal muscle oxidative metabolism.
QO

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This hypothesis was only in part confirmed in a subsequent
study.48 These authors determined by NIRS, by utilizing the
ICG dye dilution method,98 intercostal, and quadriceps muscles_ m in COPD patients breathing room air or a normoxic heliumQ_ 2p;max ,O2 mixture. During cycling exercise at 75% of VO_ m in both intercoshelium-O2 was associated with increases in Q
tal and quadriceps muscles, together with an increased CaO2
and with signs of enhanced exercise tolerance. The authors
hypothesized that changes in lung mechanics
induced by:helium-O2 likely increased stroke volume, Q and systemic_ 2 . A subsequent study187 went a step further. These authors
QO
subdivided COPD patients into dynamic “hyperinflators” and_ m by the ICG“nonhyperinflators,” measured quadriceps Q_ 2m . Helium-O2 increased exercise
method98 and calculated QO_ 2m in both groups, but at least in part different
tolerance and QO
mechanisms were involved: in hyperinflators, helium-O2_ 2m in the presence of no changes inincreased
CaO2 and QO::
Q; in nonhyperinflators, helium-O2 improved Q, vascular con_ 2m . These studies nicely demonstrate that, also
ductance, and QO
in COPD patients, NIRS can give valuable insights into themechanisms regulating the integrated responses to conditionsof increased O2 demand.
Also, COPD patients showed the [deoxy(Hb+Mb)] overshoot during constant work rate exercise142 and a slower reoxygenation kinetics during the recovery following exercise.188,189
Following 6 weeks of high-intensity training, the reoxygenation
kinetics became faster, in association with an increased citratesynthase activity and an increased exercise tolerance.188

5.4

Metabolic Myopathies and Muscular
Dystrophies

Since NIRS oxygenation indices reflect skeletal muscle fractional O2 extraction, they appear to be ideally suited to identify
and evaluate the impairment of oxidative metabolism in patientswith mitochondrial myopathies (MM) and myophosphorylase
deficiency (McArdle disease, McA), in whom the incapacity
to increase skeletal muscle fractional O2 extraction represents
a key pathophysiological mechanism (see the discussion in
Grassi et al.110). Following the pioneering work by Bank andChance,78 Grassi et al.,110 using NIRS, measured [deoxy(Hb+Mb)] at exhaustion (½deoxyðHb þ MbÞpeak ) during an incremental exercise test to obtain the maximal capacity of skeletal
muscle fractional O2 extraction in MM and McA patients. The
values were significantly lower than those of controls, and they
_ 2p;peak ,110 a close approximation
were linearly correlated with VO_ 2p;max ; they also were inversely related to the time-constantof VO_ 2p kinetics,190 another classical variable of functionalof VO
evaluation of skeletal muscle oxidative metabolism.3,4,10 The
most severely impaired patients could not increase their fractional O2 extraction during exercise compared to the values
obtained at rest, confirming observations obtained in previous
studies by invasive measurements (see the discussion in
Grassi et al.110). Moreover, ½deoxyðHb þ MbÞpeak values
obtained in MM and McA patients were substantially lower
than those obtained in patients with similar signs and symptoms
but with a negative biopsy for any known metabolic
myopathy.110 In other words, NIRS allowed one to identify
and quantify the metabolic impairment in MM and McA
patients and could also offer diagnostic clues, for example, inthe selection of patients on whom to perform a muscle biopsy.
Markedly lower [deoxy(Hb+Mb)] values at submaximal
work rates were observed in MM and McA patients.110,140
Journal of Biomedical Optics

Lower [deoxy(Hb+Mb)] values at the same submaximal workrate, versus controls, were also observed in MM during incremental handgrip exercise.191 These data suggest an excess in_ 2m for the same VO_ 2m (and work rate), which represents
QO
one of the key pathophysiological mechanisms of the diseases.
MM and McA patients also showed a marked transient[deoxy(Hb+Mb)] overshoot during constant work rate
exercise.140 In McA, the overshoot disappeared in the presenceof the “second wind” phenomenon, a sudden decrease in heart
rate and perceived exertion occurring in these patients after a
few minutes of exercise, or during a second exercise precededby a few minutes by a prior exercise.140 The second wind phenomenon is thought to derive from an enhanced sympathoadrenal response, which would allow the utilization of glucose
deriving from blood or extramuscular sources and of freefatty acids, thereby bypassing the metabolic impairment (theinability to breakdown intramuscular glycogen) at the basisof the disease (see the discussion in Porcelli et al.140). In
other words, in McA patients, NIRS confirmed the mechanism(sudden enhancement of oxidative metabolism) at the basis of
the second wind phenomenon.
Another glycolytic defect leading to a decreased capacity toincrease fractional O2 extraction during exercise is muscle-type
phosphofructokinase (M-PFK) deficiency. The impairment has
been demonstrated by NIRS in humans70 and in dogs.192½deoxyðHb þ MbÞpeak was also determined by Marzorati
et al.193 in a small population of patients with the adultform of Pompe disease (glycogen storage disease type II,
due to the deficiency of the lysosomal enzyme acid maltase),
before and after 12 months of enzyme replacement therapy.
Following enzyme replacement therapy, the authors observedthat ½deoxyðHb þ MbÞpeak slightly increased in some of thepatients.
In patients with Friedreich’s ataxia, the genetic defectdecreases the expression of the protein frataxin, localized in
mitochondria. The resulting impairment of mitochondrial function and oxidative metabolism has been identified at the skeletal
muscle level by 31 P-MRS (see the discussion in Lynch194).
These authors observed by NIRS a slower reoxygenation
kinetics following exercise, and the degree of impairment of
this variable correlated with the length of the triplet GAA repeat,a genetic measure associated with the age of onset of thedisease.194
Quaresima and Ferrari195 first investigated by NIRS
quadriceps muscle oxygenation in patients with Duchenne
muscular dystrophy (DMD). An insufficient increase in_ 2m with respect to VO_ 2m occurs in these patients.196 In
QO
other words, the functional defect is the opposite of that characterizing MM and McA patients (see above). The dystrophin
deficiency results in a loss of sarcolemmal neuronal NO. NO
plays a key role in the control of muscle blood flow during
exercise, by blunting the vasoconstrictor response to
α-adrenergic stimulation.123,124 In DMD patients, the impaired
NO synthesis would impair the intramuscular matching of_ 2m versus VO_ 2m , leading to an increased O2 extraction,
QO
which has indeed been demonstrated by NIRS.196 An
enhanced O2 extraction has been observed197 also in patientswith Becker muscular dystrophy, a milder form of dystrophy
in which the intramuscular NO impairment is also present.
In these patients, the enhanced O2 extraction was associatedwith the functional impairment. An enhanced O2 extraction,on the other hand, has not been described in patients with

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limb-girdle muscular dystrophy, in whom neuronal NO synthase is not impaired.196
Caliandro et al.198 could differentiate, by utilizing arterial
occlusion testing in postocclusion hyperemia, patients with dermatomyositis and inclusion-body myositis from healthy controls; the differentiation was not possible for patients withpolymyositis. A mitochondrial dysfunction, evaluated by the_ 2m (byrecovery kinetics of [PCr] (by 31 P-MRS) and VO
NIRS), was observed in patients with amyotrophic lateralsclerosis.199

5.5

Other Diseases

Other patients populations characterized by a skeletal muscle
oxidative impairment, which was identified by NIRS by the_ 2m following voluntary
analysis of the rate of decrease in VOor electrically stimulated contractions,94,200 are those with cystic
fibrosis201 or spinal cord injuries.202 In a case report, Ryanet al.200 described a threefold increase of the rate of decrease_ 2m (indicating a substantial improvement of skeletal
in VO
muscle oxidative metabolism) in a patient with a spinal cord
injury who underwent 12 weeks of endurance training, conducted by neuromuscular electrical stimulation.
Malenfant et al.203 observed by NIRS, in patients with pulmonary arterial hypertension, markedly higher [deoxy(Hb+Mb)] during submaximal exercise at the same absolute work
rate versus controls, indicating a markedly higher vastus lateralis fractional O2 extraction. The data suggest an impaired_ 2m in these patients, which would be compatible with the
QOlower capillary density observed in the same muscle of theother leg. The data would also be compatible with the increases
in endothelin-1 levels and with the decreases in NO and prostaglandins. Interestingly, hyperoxic breathing, while normalizing SO2a , did not substantially affect the [deoxy(Hb+Mb)]_ 2m deriving from
response, confirming an impairment in QO
the structural and functional impairments mentioned above.
In patients with fibromyalgia, Shang et al.204 combined theutilization of a commercially available NIR oximeter with acustom-built NIR diffuse correlation spectroscopy (DCS) instru_ m measurements. Experiments were conducted
ment for tissue Q
during fatiguing knee extensors isometric contractions and aforearm arterial occlusion protocol. From the O2 extraction_ m data, VO_ 2m was calculated (see also below). The
and Q
patients showed, versus the controls, a lower fractional O2
extraction and a slower reoxygenation kinetics.
Muscle NIRS measurement is an important tool in identifying high-risk patients in septic and hemorrhagic shock, by offering a continuous and noninvasive monitoring of tissue
hypoperfusion.205,206 According to these authors, during active
shock resuscitation changes in skeletal muscle oxygenation levels are correlated with changes in O2 delivery, base deficit, andblood lactate levels. A drop in muscle oxygenation levels predicts death and multiorgan failure, and may be helpful in making
critical clinical decisions.

6

Instrumentation Advancements

The present section is aimed at briefly reporting the advancements made in the last few years in the field of NIRS instrumentations suitable for muscle studies and at mentioning the future
directions of the development/utilization of these NIRS devices.
The main commercial NIRS oximeters for muscle studies,with the related key features and measurable parameters, arelisted in Table 1. Since 2011 (when a homologous table was
Journal of Biomedical Optics

published in the review by Ferrari et al.24), different batteryoperated portable/wireless muscle oximeters have been commercialized. The CW-NIRS portable/wearable systems, withwireless data transmission, are reported in Table 2. The details
about each system can be found in the related company’s
websites.
Three different NIRS techniques are utilized in the field ofmuscle oximetry. Each of them is based on a specific type of
illumination: (1) the most commonly used CW instruments,
based on constant illumination of the tissue, simply measure
the attenuation of light through the tissues; (2) FD instruments,
illuminating the tissues with intensity-modulated light, measureboth the attenuation and the phase delay of the emerging light;and (3) TD instruments, illuminating the tissue with short pulsesof light (few picoseconds), detect the temporal broadening of thepulse after propagation through the tissues. The quantitation of
the NIRS measurable parameters depends on the adopted NIRS
technology.
Different NIRS methods using the CW modality have beendeveloped to measure muscle O2 saturation (SO2m , %). The
SO2m measurement assures an accurate quantitation of the oxygenation changes occurring at the skeletal muscle level. The
CW-based systems offer the advantages of low-cost and easytransportability.
In ascending order, CW-, FD-, and TD-based instruments
require increased cost and technological complexity. On theother hand, FD and TD techniques offer a better depth discrimination than that achieved by CW instruments and allow theabsolute characterization of the tissue optical properties (absorption and reduced scattering coefficients), from which it ispossible to retrieve absolute concentration values of [oxy(Hb+Mb)] and [deoxy(Hb+Mb)], and the derived parameter[total(Hb+Mb)]. The available CW-NIRS instruments do not
determine reduced scattering coefficient and circumvent the
problem by assuming that the coefficient is substantially constant during rest and exercise. This assumption has been
criticized.207 The issue has also been discussed in the review
by Ferrari et al.24
Most of the NIRS instruments reported in Table 1 are represented by two to six channels and CW brain oximeters utilizedalso for physiological/pathophysiological muscle studies. These
oximeters are the most commonly utilized NIRS instruments
because they are of relatively low/moderate cost and transportable. Muscle NIRS instrumentations are characterized by a relatively high temporal resolution; the sampling rate is up to
100 Hz in some CW systems, 50 Hz in the only commercial
FD system, and 0.2 Hz in the first TD oximeter with CE
mark, commercialized in 2015 (Table 1). An original muscle
oximeter has been very recently introduced by Reflectance
Medical; the instrument collects muscle spectra and calculates
SO2m as well as pHm .
The sector of wearable health technology is gaining increasing
interest. The use of low cost wearable devices or wearable biosensors, which allow a constant monitoring of physiological signals, is essential for the advancement of both the diagnosis andthe treatment of diseases, as well as for monitoring active life
style. Very recently, a textile NIRS system has been utilized
for monitoring arterial O2 saturation (measured by pulse oximetry) and muscle oxygenation changes.208 Wearable wireless fingertip pulse oximetry devices, for arterial O2 saturation
measurement, are already available for smart phones. In addition,a wireless and smartphone controllable NIRS system (DynaSense

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Inc., Japan) has been very recently presented.209 Table 2 includes
also two wireless systems that are waterproof, then allowing
muscle oxygenation measurements in swimmers.210
For a better understanding of skeletal muscle physiology,
muscle oxygenation data should be ideally integrated with
regional blood flow measurements. Direct and indirect NIRS
_ m 20,96 have been discussed above. A
methods to measure Q
novel technique, known as DCS, has been recently developedfor quantifying relative changes in microvascular regional
blood flow. Basically, DCS uses coherent NIR light to penetrate
deep tissues and measures speckle fluctuations of the diffuse
light that are sensitive to the motions of red blood cells intissues.211,212 The well validated DCS provides a portable, noninvasive, and inexpensive alternative for measuring microvascular blood flow. A recent review article has described the_ m during
progresses made in the field of DCS for assessing Q
213
dynamic exercise. This technology allows also the estimationof skeletal muscle oxygenation levels. So far, two DCS-NIRS
systems have already been commercially available: αιμαFloMo (Hemophotonics S.L., Spain) and MetaOx (ISS,_ m, while the latest
United States). The former measures only Q
provides concomitant measurements of SO2m and absolute [oxy(Hb+Mb)] and [deoxy(Hb+Mb)] by FD-NIRS. The method has
been utilized on the head of newborns and on the skeletal
muscles of adults. Giovannella et al.214 have integrated theαιμα-FloMo with a TD-NIRS prototype (TRS20, Hamamatsu_ m and absolute [total(Hb+Photonics, Japan) to measure QMb)] during quadriceps exercise. A different approach for meas_ m has been utilized by Ornim Inc. (United States), which
uring Qhas developed the brain/muscle oximeter “CerOx 3210F” using
NIRS and weak acoustic beams to identify light emerging fromdeep tissue layers.
Many sophisticated multichannel CW-NIRS instrumentations, dedicated to brain cortex two-dimensional (2-D) imaging(topography), are commercially available and largely utilized indifferent areas in the field of neuroscience.25,215 Unfortunately,
the probes they are equipped with cannot be utilized for skeletal
muscle 2-D imaging studies. In 2015, an eight-measurement
point wireless system for 2-D brain/muscle measurements has
been commercialized (Table 2). In the framework of a
National Aeronautics and Space Administration (NASA)project, a 64-measurement point wireless NIRS system has
been developed and integrated with simultaneous electrocardiography and actigraphy monitoring, in order to record 2-D
muscle imaging data up to several days.216 Although a threedimensional (3-D) NIR imaging (tomography) of the humanforearm, based on TD techniques, had been proposed217 almost
15 years ago, no further consistent progresses in this field havebeen made. Recently, cross-sectional 2-D images of the foot[total(Hb+Mb)], using a multichannel 3-D CW system during
thigh cuff occlusion, were reported.218
The state-of-the-art of TD-NIRS has been recently
reviewed.219 Many progresses have been done in the last 2
years that open a new way to compact, low-cost, and highperformance TD devices for diffuse muscle optical imaging
and spectroscopy. The new frontiers in TD diffuse optics arecovered by an article of this Special Section. 220 Briefly, consistent progresses are expected concerning noncontact scanning
2-D imaging systems for TD-NIRS based on a null source–
detector separation.221–223 These systems, already tested in thehuman forearm, utilize new fast detectors for extreme depth penetration and sensitivity.224,225
Journal of Biomedical Optics

7

Conclusions

Researchers should always be critical and careful about the technologies and methods they utilize, but within reasonable limits.
Many exercise physiologists, in our opinion, have been excessively critical about NIRS in the past. The method has important
limitations but presents significant advantages compared toother methods. Skeletal muscle fractional O2 extraction, themain variable that can be noninvasively evaluated by NIRS,
can yield relevant information on key physiological and pathophysiological mechanisms, relevant in the evaluation of exercise
performance and exercise tolerance in healthy subjects and inpatients.
To put the method in a broader perspective it should be recognized, for example, that the BOLD phenomenon,226 as obtainedby functional magnetic resonance imaging, a functional neuroimaging technique which has profoundly revolutionized neuroscience with applications ranging from normal brain development,
aging, brain disorders and diseases, is conceptually very similar to
the fractional O2 extraction variable determined by NIRS in skeletal muscle. Both variables, indeed, substantially reflect changes
in tissue oxygenation resulting from the dynamic balance
between tissue O2 consumption and O2 supply.
The recent diffusion of apparently easy-to-use portable NIRS
instruments had led to the proliferation of studies, sometimes of
dubious quality. An example is represented by studies, whichclaim to obtain absolute oxygenation indices when the instrumentation they employ does not allow this. The authors must
be aware of the limitations of the method and should always
have a clear and sound hypothesis to test. However, in theright hands (a good physiological background is mandatory),
NIRS can offer insights into key physiological and pathophysiological adaptations to conditions of increased O2 needs, often
involving, in an integrated manner, different organs of the
body. In terms of patients’ evaluation, NIRS allows to determine
the evolution of functional impairments of skeletal muscle oxidative metabolism with time, to identify correlations withclinical symptoms, to evaluate the effects of therapeutic or rehabilitative interventions, and to gain pathophysiological and diagnostic insights.

Acknowledgments
The authors would like to thank Rosanna De Carolis for thetechnical support.

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Bruno Grassi, received his MD and PhD degrees and now is a professor of physiology in the Medical School of the University of Udine(Italy), where he is head of the Exercise Physiology Laboratory. His
research interests are in the fields of exercise and environmental
physiology, skeletal muscle bioenergetics, with special reference tooxidative metabolism, in health and disease. He is the author of
112 scientific publications on international peer-reviewed journals
and of 25 chapters in books.
Valentina Quaresima received her PhD and now is an associate professor of biochemistry at L’Aquila University, Italy. She has pioneered
clinical applications of NpIRS of the adult and infant muscle/brain andhas given substantial contributions to wider NIRS scientific community. Since 1993, she has been involved in the study of localized measurements of muscular and cerebral oxygenation in humans bydifferent NIRS techniques. She is the author of 100 scientific publications on international peer-reviewed journals.

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