Phil. Trans. R. Soc. A (2011) 369, 1–14
doi:10.1098/rsta.2011.0230

REVIEW

The use of near-infrared spectroscopy inunderstanding skeletal muscle physiology:
recent developments
B Y M ARCO F ERRARI1, *, M AKII M UTHALIB2 AND V ALENTINA Q UARESIMA1
1 Department of Health Sciences, University of L’Aquila, L’Aquila, Italy
2 School of Human Movement Studies and Movement Neuroscience Program,

Institute of Health and Biomedical Innovation, Queensland Universityof Technology, Brisbane, Queensland, Australia
This article provides a snapshot of muscle near-infrared spectroscopy (NIRS) at the endof 2010 summarizing the recent literature, offering the present status and perspectivesof the NIRS instrumentation and methods, describing the main NIRS studies on skeletal
muscle physiology, posing open questions and outlining future directions. So far, different
NIRS techniques (e.g. continuous-wave (CW) and spatially, time- and frequency-resolved
spectroscopy) have been used for measuring muscle oxygenation during exercise. In
the last four years, approximately 160 muscle NIRS articles have been published on
different physiological aspects (primarily muscle oxygenation and haemodynamics) of
several upper- and lower-limb muscle groups investigated by using mainly two-channel
CW and spatially resolved spectroscopy commercial instruments. Unfortunately, in only
15 of these studies were the advantages of using multi-channel instruments exploited.
There are still several open questions in the application of NIRS in muscle studies:(i) whether NIRS can be used in subjects with a large fat layer; (ii) the contribution of
myoglobin desaturation to the NIRS signal during exercise; (iii) the effect of scatteringchanges during exercise; and (iv) the effect of changes in skin perfusion, particularly
during prolonged exercise. Recommendations for instrumentation advancements andfuture muscle NIRS studies are provided.
Keywords: skeletal muscle; near-infrared spectroscopy; near-infrared imaging;muscle oxygenation; muscle oxy-haemoglobin saturation; muscle metabolism

1. Introduction
Since the end of the 1980s, near-infrared spectroscopy (NIRS) has been used toinvestigate local muscle oxidative metabolism at rest and during different exercise
modalities. The unique advantage of using NIRS is that, when proper care is*Author for correspondence (marco.ferrari@univaq.it).
One contribution of 20 to a Theo Murphy Meeting Issue ‘Illuminating the future of biomedical
optics’.

1This journal is © 2011 The Royal Society

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M. Ferrari et al.

taken to minimize movement artefacts, it can yield acceptable signal-to-noise
ratios during exercise. Ferrari et al. [1] reviewed the ﬁrst decade of NIRS muscle
studies at the dedicated 1996 Royal Society Discussion Meeting. In the following
years, several other reviews covered different aspects of muscle NIRS researchsuch as sports science and clinical medicine [2,3]. Hamaoka et al. [4] reviewed themethodological issues of NIRS and near-infrared (NIR) imaging for monitoring
muscle oxygenation and haemodynamics in healthy and diseased humans. The
234 references quoted in that review article witness not only the evolution of the
NIRS/NIR imaging techniques, but also their signiﬁcant applications in exercise
physiology and clinical medicine.
A search on the databases PubMed, Scopus and Web of Science was performed
using the keywords: ‘near-infrared’, ‘near-infrared oximetry’, ‘muscle oxygenation’and ‘optical imaging’. From 2007 up to the end of 2010, approximately 160
articles related to muscle NIRS studies (excluding the clinical related studies)were published on different aspects of muscle physiology (primarily muscle
oxygenation and haemoglobin (Hb) volume) of several upper and lower limb
muscle groups investigated by using mainly one-/two-channel commercial NIRS
instruments. Only 15 of these studies exploited the advantages linked to the useof multi-channel instruments.
Considering that the number of laboratories and articles related to theapplication of NIRS in muscle studies is increasing, most muscle physiology
researchers would agree with the assertion that muscle NIRS research is
progressing. The technology and methodology of NIRS instruments have shown
advances in robustness and sophistication. This article is an attempt to provide
a snapshot of muscle NIRS at the end of 2010 summarizing the recent literature,offering the present status and perspectives of the NIRS instrumentation andmethods, the main ﬁelds of applications for studying skeletal muscle physiology,
the open questions and future directions.

2. Present status and perspectives of the near-infrared spectroscopy muscle
instrumentation and methods(a) Commercial near-infrared spectroscopy systems used in muscle studies
The different types of NIRS instruments with the related key features, advantages
and disadvantages have been previously reviewed in detail [5]. Brieﬂy, threedifferent NIRS techniques are used, each based on a speciﬁc illumination type:(i) the continuous-wave (CW) modality, based on constant illumination of
the tissue, simply measures the attenuation of light through the tissues; (ii)frequency-domain (FD) instruments, illuminating the tissues with intensitymodulated light, measure both the attenuation and the phase shift of theemerging light; and (iii) the time-domain (TD) technique, illuminating the tissues
with short pulses of light, detects the shape of the pulse 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 been developed to measure the oxy-Hb (O2 Hb) saturationof the muscle (SmO2 , %), one being the spatially resolved spectroscopy(SRS) method, which is the most widely used oximetry approach. The SmO2
measurement ensures an accurate quantitation of the oxygenation changes
Phil. Trans. R. Soc. A (2011)

Review. NIRS and muscle oxygenation physiology

3

occurring at the muscle level. SmO2 reﬂects the dynamic balance between
O2 supply and O2 consumption in the investigated muscle volume. CWbased systems offer the advantages of low cost and easy transportability. In
ascending order, CW-, FD- and TD-based instruments require increased cost
and technological complexity. On the other hand, only FD and TD techniques
offer the absolute characterization of the tissue optical properties (absorption
and reduced-scattering coefﬁcients), from which it is possible to retrieve absoluteconcentration values of O2 Hb and deoxy-Hb (HHb), and a derived parameter
total-Hb (tHb = O2 Hb + HHb). Moreover, a scaled absolute value for tHb can
be obtained by the SRS method. NIRS methodology is characterized by arelatively high temporal resolution (sampling rate up to 100 Hz with CWand FD-based instruments, and around 5 Hz with TD-based instruments)
enabling the measurement of the time course of changes in SmO2 during brief(2–4 s) leg press exercise [6] or even during a single pedal cycle of a cycling
exercise [7].
Table 1 reports the main current commercial NIRS instruments used in muscle
studies. The details of each system can be found in the relevant company’s
website. Most of the instruments are represented by two-channel brain oximeters
used also in muscle studies. These oximeters are the most commonly used
instruments for muscle studies because they are of relatively low/moderatecost and transportable. The NIRS instrumentation can be miniaturized andeven made wireless. Unfortunately, no standardization is yet available for NIRS
instrumentation. In particular: (i) the operating range (i.e. the interval within
which the instrument works reliably) of the oximeters should be recognized andindicated and (ii) comparison between oximeters at rest and during dynamic
exercise should be performed.
Recent advances in NIRS technology have included the introduction of
NIR multi-channel systems (which use arrays of multiple NIR sources anddetectors) that, by simultaneously collecting data from multiple muscle regions,avoid variations caused by position-dependent differences in muscle oxygenation(a problem inherent with single-location measurements). Multi-channel NIRS
systems provide only topographic NIR images (i.e. two-dimensional images)
and, in this article, they are referred to as ‘imagers’. Typical depth sensitivityof the CW-based imagers is approximately 1.5 cm and the spatial resolution
is limited to approximately 1 cm. TD- and FD-based imagers are the mostpowerful tools to investigate the spatial and temporal proﬁles of the absolutechanges in SmO2 and tHb during exercise; however, the signal-to-noise ratio of
measurements is low. Although prototypes of muscle NIR imagers have been
available since the end of the 1990s, NIR imaging has been rarely used in muscle
studies probably owing to its relatively high cost and complexity compared with
one-two-channel oximeters.

(b) New methods and signal interpretability
The muscle parameters measurable directly and indirectly by NIRS andNIR imaging have been reviewed by Wolf et al. [5]. Muscle NIRS parametermeasurements are repeatable and reproducible during elbow ﬂexor exercise [8]and cycling exercise [9].
Phil. Trans. R. Soc. A (2011)

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M. Ferrari et al.

Table 1. Main current commercial near-infrared spectroscopy systems used in muscle studies.
CW, continuous-wave spectroscopy; FD, frequency-domain spectroscopy; TD, time-domain
spectroscopy; u.r., upon request.

instrument
photometers
BOM-L1 TRa
PocketNIRS Duoa,boximeters
Astem Hb-12ca−c
InSpectra 650d,e
INVOS 5100Cd,f
NIMOc
NIRO-200NXfOXYMON-II Af
OxiplexTS
PortaMonb,gTRS-20a,f

techniquecompany

websiteOmegawave,
Japan
Hamamatsu,
Japanwww.omegawave.co.jp

single-distance
CW
single-distance
CW

1

multi-distance
CW
multi-distance
CW
multi-distance
CW
multi-distance
CW
multi-distance
CW
multi-distance
CW
multi-distance FD
multi-distance
CW
TD

2

Astem, Japan

www.astem-jp.com

1

Hutchinson, USA

4

Somanetics, USA

www.htbiomeasurement.
com
www.somanetics.com

1–4

Nirox, Italy

www.nimoworld.com

2

Hamamatsu,
Japan
Artinis, The
Netherlands
ISS, USA
Artinis, The
Netherlands
Hamamatsu,
Japanwww.hamamatsu.com

multi-channel systems h
Dynot
CW
CW
FOIRE-3000d
Imagent
FD
NIRO-200CWNIRS2 CE, CW6OXYMON MkIII

no. ofchannels

CW
CW

2

2
2
1
2

u.r.
NIRx, USA52
Shimadzu, Japan
up to 128 ISS, USA
10
Hamamatsu,Japan
up to 176 TechEn, USA
up to 96 Artinis, The
Netherlands

www.hamamatsu.com

www.artinis.com
www.iss.com
www.artinis.com
www.hamamatsu.com

www.nirx.net
www.med.shimadzu.co.jp
www.iss.com
www.hamamatsu.com
www.nirsoptix.com
www.artinis.com

a Commercially available only in Japan.
b Wireless system.
c Oximeter with fat-layer compensation in real time.
d US Food and Drug Administration’s approval.
e Only for the thenar muscle.
f Brain oximeter also used in muscle studies.
g An accelerometer is available on request.
h Multi-channel systems have been developed for cortical activation studies and scarcely used on

muscle.

Phil. Trans. R. Soc. A (2011)

5

Review. NIRS and muscle oxygenation physiology
(a)

(b)

torque (Nm)

60
40
20
0

ΔSmO2 (%)

0

ΔSmO2max

ΔSmO2max

20
ΔSmO2slope

ΔSmO21/2DT

ΔSmO21/2RT–20
ΔSmO21/2RT

–40
–60

ΔSmO2TD
ΔSmO2A

ΔSmO2minΔSmO2tΔSmO2minΔtHb (Mcm)

200
0

–200
–400
–600

ΔtHbmax
ΔtHbmean–5 0 5 10 15 20 25 30 35 40 45 50 55 60
time (s)ΔtHbmax
ΔtHbmean
0 10 20 30 40 50 60 70 80 90 100 110 120
time (s)Figure 1. Typical changes in elbow ﬂexor torque and biceps brachii muscle oxyhaemoglobin
saturation (DSmO2 ) and total haemoglobin volume (DtHb) during a 10 s sustained (a) and 30
repeated (b) isometric exercise task at 100% maximal voluntary contraction. The vertical dottedlines delimit the duration of the exercise. DSmO2slope , DSmO2 desaturation slope; DSmO2 min ,minimum DSmO2 amplitude; DSmO2 max , maximum DSmO2 amplitude; DSmO21/2RT , DSmO2
half resaturation time; DSmO21/2DT , DSmO2 half desaturation time; DtHbmean , DtHb mean
decrease; DtHbmax , DtHb maximum increase. Inset (b): SmO2 kinetic analysis using nonlinear
regression analysis. SmO2TD , initial time delay; SmO2 t, exponential time constant; DSmO2A ,DSmO2 desaturation amplitude.

An example of the typical changes in biceps brachii SmO2 and tHb (measured
by a two-channel oximeter) during a 10 s sustained and 30 repeated isometric
exercise of the elbow ﬂexors at maximal voluntary contraction is shown inﬁgure 1. During the sustained contraction (ﬁgure 1a), DSmO2 desaturation
slope (DSmO2slope ) is the negative slope of the least-squared regression line ofDSmO2 during the contraction phase; a higher DSmO2slope would represent agreater muscle O2 demand, and consequently a greater energy consumption[8,10]. DSmO2 minimum amplitude (DSmO2 min ) is the difference between theminimum SmO2 value reached during the contraction phase and SmO2 baseline;
lower DSmO2 min values represent greater O2 demand relative to O2 supply[6,10]. DSmO2 maximum amplitude (DSmO2 max ) is the difference between the
maximum DSmO2 value reached during the relaxation phase and SmO2 baseline;
higher DSmO2max values indicate increased O2 supply relative to O2 demand [6].
DSmO2 half-recovery time (DSmO21/2RT ) is the time to reach 50 per cent of thedifference between DSmO2 at the end of the contraction phase and DSmO2max inPhil. Trans. R. Soc. A (2011)

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M. Ferrari et al.

the recovery period; a faster DSmO21/2RT is related to muscle oxidative capacity[8]. Mean decrease in DtHb (DtHbmean ) is the difference between the average
DtHb value during the contraction phase and tHb baseline; greater decreases andrelatively stable DtHbmean values during the contraction phase would indicate
blood ﬂow/O2 supply occlusion owing to increased intramuscular pressure of thecontraction [11]. Maximum increase in DtHb (DtHbmax ) is the difference between
the maximum DtHb value reached during the relaxation phase and tHb baseline;
higher DtHbmax values would indicate a greater increase in blood volume/ﬂow [6].
During dynamic exercise (ﬁgure 1b), DSmO2 min is the difference between
the minimum DSmO2 min value reached during the 30 contractions and SmO2
baseline; a given DSmO2 min value represents the dynamic balance of O2 supply
by the microcirculation and O2 consumption by the mitochondria [8]. DSmO2
desaturation time (DSmO21/2DT ) is the time difference between contraction onset
until DSmO2 reaches 50 per cent of the difference value between SmO2 baseline
and DSmO2 min ; a longer duration DSmO21/2DT for a similar DSmO2 min would
represent a slower desaturation rate, indicating that O2 demand is better matchedby O2 supply. DtHbmean is the difference between the average of the minimum
DtHb amplitude reached during the 30 contractions and tHb baseline; a given
DtHbmean value would indicate the level of blood volume or blood ﬂow/O2 supply
over the exercise duration [11].
The kinetics of muscle oxygenation can be modelled by nonlinear regression
using least-squares techniques [12], and the kinetics of SmO2 is represented by
the following formula:
SmO2 (t) = SmO2base − SmO2A (1 − e−(t−SmO2TD )/SmO2 t ),where SmO2base represents the resting SmO2 baseline value. The curve ﬁt forthe ﬁrst 30 s of exercise is shown in the inset of ﬁgure 1. After the onset of
exercise (represented by the ﬁrst dashed line), an initial time delay (SmO2TD ),followed by a rapid desaturation in SmO2 , is described with an exponential
time constant (SmO2 t) representing the time to reach 63 per cent of thedesaturation response. SmO2 desaturation amplitude (SmO2A ) is the difference
between the SmO2base and the nadir of SmO2 . The SmO2 mean response time
(SmO2MRT = SmO2TD + SmO2 t) can be calculated to provide a description of theoverall time course for muscle desaturation during the rest–exercise transition.
A faster SmO2MRT would indicate that O2 consumption is not sufﬁciently being
matched by O2 supply, and conversely, a slower SmO2MRT would suggest that O2
consumption is sufﬁciently being matched by O2 supply [12].
Many studies have evaluated the kinetics of muscle oxygenation using CW–
SRS-based oximeters and have drawn their physiological conclusions only on thebasis of the interpretation of the changes in HHb. Several arguments have beenraised to support [13] or refute [14] this choice, because these oximeters provide
SmO2 values that are independent of the pathlength of the NIR photons in themuscle tissue and, unlike HHb, are not so sensitive to the optical coupling andto the presence of superﬁcial tissue layers. Taking into account the advantages
offered in using CW–SRS-based oximeters, the physiological conclusions should
be drawn essentially on the basis of SmO2 results, and reporting tHb data.
Among the most recent methods, Leung et al. [15] determined SmO2 during
cycling exercise measuring the rhythmical changes of the NIR signal or ‘cyclic
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Review. NIRS and muscle oxygenation physiology

7

SmO2 ’. Binzoni et al. [7] further developed Leung’s method by using the ‘cyclic’signal embedded in the NIR signal to derive instantaneous SmO2 and muscle O2
consumption.

(c) Advanced near-infrared imaging technologies and multi-modal
spectroscopy/imaging
Among the advanced commercial NIR technologies used for muscle studies
in the last 4 years, it is signiﬁcant to mention the following three approaches:(i) CW–SRS imaging, (ii) TD imaging, and (iii) wireless CW imaging. Kek
et al. [16] developed a portable muscle NIR imager, based on CW–SRS, whichis capable of measuring SmO2 over 32 measurement points (with a fat-layer
correction algorithm) during quadriceps dynamic exercise. TD muscle imaging
instruments have the advantage of improving the depth sensitivity by exploiting
the temporal information of photon migration through tissues (i.e. early photons
primarily representing superﬁcial layers and late photons representing deeper
muscle layers). The Politecnico of Milan (Italy) developed a two-wavelength
TD imager with a sampling time of 6 Hz and allowing up to 32 measurement
points [17].
NIR imaging systems use cables to connect the sensor that is attached to thesubject to the acquisition electronics. These cables are disturbing and can cause
motion artefacts by dislocating the sensor if care is not taken to secure the cables
properly. For these reasons, a lightweight wireless NIR imager was developed [18].
In the last 20 years, muscle NIRS has been employed also in combination witha large variety of other non-invasive and invasive methodologies for evaluating
pathophysiological changes in peripheral muscle (for a review see Hamaoka
et al. [4]). A recent study demonstrated the usefulness of the combination of
NIRS with nuclear magnetic resonance (NMR) techniques for obtaining robust
information regarding muscle oxidative metabolism and haemodynamics [19].

3. Main ﬁelds of near-infrared spectroscopy applications for studying skeletal
muscle physiology
So far, only about 20 out of the approximately 600 skeletal muscles havebeen investigated by NIRS. Speciﬁcally, lower limb muscles (i.e. biceps femoris,
gastrocnemius, rectus femoris, tibialis anterior, vastus lateralis, vastus medialis)
were studied during diverse conditions (cycling, upon electrical stimulation,
during knee extension exercise, leg press exercise, plantar ﬂexion exercise,running, squatting, Wingate test), and upper limb muscles (i.e. biceps brachii,brachioradialis, deltoid, forearm ﬂexors, triceps brachii) during diverse conditions(arm abduction exercise, bench press exercise, cycling, elbow ﬂexion exercise,upon electrical stimulation, handgrip, rowing, Wingate test). The trunk muscles(i.e. erector spinae, intercostal, multiﬁdus, paravertebral, serratus anterior) werealso investigated during cycling, back extension and bending forward.
So far, NIRS has been applied for studying exercise-induced muscle
damage [11,20], ergonomics/biomechanics [21], heterogeneity of muscle O2
supply/demand [12,22], muscle activation [11,23], priming exercise [19,24],
respiratory muscle blood ﬂow/fatigue [25,26], the role of the brain in muscle
fatigue [27,28], the time course of oxidative metabolism [6,29] and the effect ofPhil. Trans. R. Soc. A (2011)

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M. Ferrari et al.

exercise training [30]. Owing to the restriction of the allocated space, this review
article neglects an in-depth discussion of the 160 studies published in the last
four years.
It is important to mention that among the muscle NIRS articles published in
the last four years only 15 were performed by using NIR imagers. It is envisionedthat more studies in the future will use the beneﬁts of NIR imagers to study
regional differences in muscle O2 supply–demand responses to exercise. The effects
of different motor tasks [31] and the effects of exercise/muscle fatigue [32] on bothmuscle and cortical oxygenation changes have been previously reviewed.

4. Open questions
Some of the most signiﬁcant open questions in order of importance are: (i) the
possibility to use NIRS in subjects with a large fat layer; (ii) the contributionof myoglobin (Mb) desaturation to the NIRS signal during exercise; (iii) the
effect of scattering changes during exercise on CW–NIRS muscle measurements;(iv) the effect of changes in skin perfusion on CW–NIRS muscle measurements,
particularly during prolonged exercise; and (v) the possibility to measuremuscle/interstitial pH.
It is well known that the relatively high attenuation of NIR light in muscle
measurements is owing to: (i) the two main chromophores—Hb and Mb; (ii)light scattering; and (iii) other molecules (mainly skin melanin, water, lipids ofthe adipose tissue, intra-muscle lipids and cytochrome c oxidase). The inﬂuenceof adipose tissue thickness (ATT) on light propagation in leg muscles has been
examined by several researchers. Muscle NIRS testing has been limited to slim
subjects (preferably men because they have a smaller ATT with respect towomen). Patients with diabetes, paraplegia or chronic fatigue syndrome, whohave a tendency to be obese, cannot be investigated by NIRS if ATT values
are high. At least three methods have been developed for fat-layer correction.
An algorithm capable of correcting for the inﬂuence of ATT ranging from 0 to
15 mm has been proposed [33] and it has been included in some commercial
NIRS systems (table 1). Another ATT correction algorithm and an opticalmethod for ATT monitoring have been developed [34,35]. The optical lipid signal
is a good predictor for ATT less than 16 mm. The possibility of measuring
ATT directly might be an efﬁcient alternative to the measurement of ATT by
ultrasound. Another method for measuring SmO2 has been recently reported[36]. This method uses broadband CW–NIRS after the removal of spectralinterference owing to skin, water, ATT and scattering. Unfortunately, none ofthese methods have been so far used by the most widespread commercial NIRS
system manufacturers (table 1), which strongly limits the clinical applicability ofmuscle NIRS in patients with high ATT.
There is a considerable discussion about the contribution of Hb and Mb to
the in vivo NIR signal from skeletal muscle. It is difﬁcult to differentiate Hb andMb spectra because they are very similar in the NIR range. Marcinek et al. [37],using wavelength shift analysis, showed that Hb accounts for approximately 20
per cent of the optical signal in human resting ﬁrst dorsal interosseous muscle.
However, Hb/Mb ratio in the muscle at rest is unknown, and there are few data
available on the relative kinetics of Mb and Hb desaturation during different
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Review. NIRS and muscle oxygenation physiology

9

exercise modalities. In an animal study, Mb desaturation of rat gastrocnemiousmuscle was investigated by NIRS during Hb-free medium perfusion [38]. At theonset of contraction, Mb desaturated rapidly and declined progressively withwork intensity. Proton (1 H) NMR measures muscle deoxy-Mb signal allowing theassessment of intracellular O2 availability at rest and during exercise with a time
resolution of about 8 s. In a human study, Lanza et al. [39] found that, at rest,
the tibialis anterior intramuscular O2 stores (measured by the appearance of 1 H
NMR deoxy-Mb signal during cuff occlusion) began to decrease only after 1 min,and that maximal Mb desaturation was achieved after about 6.5 min. Conversely,at rest, the intramuscular O2 stores (measured by NIRS during cuff occlusion)in different muscle groups began to decrease a few seconds after the beginningof the occlusion and maximal desaturation was achieved 4–5 min later. Duringhigh-intensity exercise, Mb typically desaturates to only 50 per cent of the levelattained during cuff occlusion, and muscle oxygenation, as measured by CW–NIRS, typically desaturates to about 90 per cent of the level attained during
the cuff occlusion. Overall, these data would suggest that the contribution of
Mb desaturation to the NIR signal during dynamic exercise is estimated to
be roughly less than 20 per cent. Combined 1 H NMR and NIRS studies could
clarify not only the issue of the contribution of Mb to the NIRS signal, but also
the relative kinetics of Mb and Hb desaturation during exercise with different
workloads.
In principle, exercise could alter the muscle tissue optical properties by causing
a change in the scattering of NIR photons. The change in scattering at different
wavelengths and the relative pathlength changes that occur during different
exercise modes in the human quadriceps and calf muscles have been studied by
TD–NIRS techniques [40,41]. It was observed that scattering coefﬁcients andpathlength in the muscle, at different wavelengths, decreased by less than 10 per
cent during the different exercise modalities. Therefore, it could be expected thatscattering and pathlength changes, occurring during exercise, would negligibly
contribute to the calculation of changes in O2 Hb, HHb and tHb measured by
CW–NIRS instruments.
Several controversial studies have been published on the effect of skin blood
ﬂow changes on muscle NIRS measurements. A recent study [42] provided
reassurance that changes in SmO2 during exercise predominantly reﬂect muscle
oxygenation, even during conditions where skin and muscle blood ﬂow were
elevated concomitantly (e.g. during prolonged, dynamic exercise).
Soller et al. [43] and previous studies showed that broadband CW–NIRS
can be used to non-invasively and continuously measure SmO2 (based on Hb
spectral features), and indirectly muscle/interstitial ﬂuid pH. Partial leastsquares regression was used to develop the multi-subject calibration equation,
which correlates NIR spectra to muscle/interstitial ﬂuid pH values obtained
invasively in the muscle of the calibration subjects. Once developed, the equation
can be applied to NIR spectra acquired from independent subjects to predictmuscle/interstitial pH. Recent studies demonstrated the feasibility of the muscle
pH estimation, the determination of H+ threshold and the relation between H+
and classical metabolic thresholds (lactate and gas exchange) during incremental
exercise [44].
A summary of the recommendations for properly using NIRS instrumentationin muscle studies is reported in table 2.
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M. Ferrari et al.

Table 2. Recommendations for near-infrared spectroscopy muscle studies. ATT, subcutaneous
adipose tissue thickness; CW, continuous-wave spectroscopy; EMG, electromyography; FD,frequency-domain spectroscopy; fMRI, functional MRI; MRI, magnetic resonance imaging; NMR,
nuclear magnetic resonance spectroscopy; SRS, spatially resolved spectroscopy; PET, positronemission tomography; TD, time-domain spectroscopy.
topic

current status

recommendations

depth sensitivity

typically approximately 1.5 cm
for 3–4 cm source–detector
distance. Measurements
restricted to superﬁcialmuscle(s)investigated muscle
volume andmeasurement points

oxygenation of large muscle
groups like the quadriceps isinvestigated by using only one
or two measurement points
often not accurately reported

use TD technologies andtomographic approaches for
improving depth sensitivity. In thecase of multi-distance CW–NIRS,
use less than 5 mm and greaterthan 30 mm for the shortest andlongest source–detector distance,
respectively
use multi-channel systems forinvestigating the spatial proﬁleof muscle/muscle groups
oxygenation
describe in detail the location,
eventually guided by ultrasoundscannerensure an adequate stable contact
between the optodes and the skinthroughout the acquisition
session. Minimize the sliding bybandage (avoiding venous
occlusion), and use NIR
transparent double-sided adhesive
tape. Monitor the pressure of the
optode on the skinmeasure ATT using skinfold
callipers, ultrasound scanner, MRI
or optical lipid signal. Eventually
perform studies only on subjects
with homogeneous ATT. Usealgorithms for ATT correction
measure skin blood ﬂow (e.g. bylaser Doppler) and/or skintemperature close to the optodein prolonged exercise
try to keep the limb movements inthe same planes in order tominimize artefacts. Artefacts
should be identiﬁed andcorrected/eliminated in the NIRS
data analysis
describe in detail the protocol forthe reproducibility/repeatabilityof the measurements

optode positioning

often not veriﬁed during and
optode–skinafter the study or notcoupling/sliding;mentioned
optode sliding owing
to sweat (especially inhairy skin) and/or
mechanical factors

adipose tissue thickness

often not reported

skin blood ﬂow changes
over the exercisingmuscleusually not measured

muscle shape changes
during exercise

usually not mentioned

exercise andexperimental set-up
description

often inaccurate(Continued.)Phil. Trans. R. Soc. A (2011)

Review. NIRS and muscle oxygenation physiology

11

Table 2. (Continued.)
topic

current status

recommendations

kinematic motor
performance

motor performance not always
monitored and controlled

absolute quantiﬁcation
value of NIRS
measures

SRS–CW-based systems provide
only SmO2 (%) quantiﬁcation

data analysis

analysis of SmO2 (%) andconcentration changes in
O2 Hb, HHb and tHb. Often
only the HHb kinetics andamplitude are analysed andreported
no standardization is available
for NIRS
instrumentation/signal
processing/data analysis
very few studies

monitor motor performance by
three-dimensional kinematic
analysis in particular for open
ﬁeld exercise
use TD- and FD-based
instrumentations for improving
sensitivity and quantitation of
NIRS parameters
analyse and report all measurable
parameters, i.e. O2 Hb, HHb, tHb,
SmO2

standardization

multi-modal studies

regulatory authorities or network of
research laboratories should
provide ‘guidelines’
integrate NIRS with MRI, fMRI,
NMR, PET, EMG, microdialysis,
Doppler blood ﬂow measurements

5. Future directions
The most exciting prospect of muscle NIRS studies for the next 20 years isthe full understanding of skeletal muscle biochemistry/physiology/pathology for
improving human healthcare, athletic performance and rehabilitation monitoring.
The major challenge to achieving this understanding might be the availabilityof a low-cost, easy-use optical wearable/wireless non-contact NIR imager for
obtaining four-dimensional SmO2 and haemodynamic (blood ﬂow and tHb)
measurements of human skeletal muscle, especially during dynamic exercise.
This ideal ‘NIR imager’ should be suitable for any application (including
general health, clinical and athletic settings), and might be an addition to thecurrent heart rate monitoring and lactate measurements during training in theﬁeld and health centre-like environments. This ideal ‘NIR imager’ should be
combined/integrated with other imaging and electrophysiological modalities forenhancing the understanding of speciﬁc muscle mechanisms in pathophysiologicalconditions.
Considering the rapid development of related technologies, it is very difﬁcult
to predict the potential advancements of muscle NIRS and NIR imaging. The
quantitative measurement of deep forearm oxygenation and tHb by a non-contact
oximeter prototype was proposed by Niwayama et al. [45].
The current typical depth sensitivity of most CW-based imagers isapproximately 1.5 cm. Therefore, a tomographic approach might provide
three-dimensional SmO2 and haemodynamic measurements. Blood ﬂow ofPhil. Trans. R. Soc. A (2011)

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M. Ferrari et al.

the superﬁcial muscles might be continuously measured by diffusing-wave
spectroscopy, a new rapidly progressing technique discussed in an accompanying
symposium paper. Although three-dimensional NIR imaging of the humanforearm, based on TD techniques, was proposed by Hillman et al. [46] almost
10 years ago, no further progress has been made to develop the technique.
In conclusion, it is foreseeable that the availability of advanced NIR imagers
would help to reﬁne the understanding of skeletal muscle oxygenation in different
pathophysiological conditions.
The research is supported in part by Hamamatsu Photonics KK, Japan. The authors would liketo thank Rosanna De Carolis for the technical support.

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