Reference Values of Regional Oxygen Saturation (rSO₂) Determined by Near-Infrared Spectroscopy (NIRS) for 18 Selected Regions of Interest (ROIs) in Young and Elderly Healthy Volunteers

Abstract

Regional oxygen saturation (rSO₂) assessed by near-infrared spectroscopy (NIRS) reflects the perfusion and metabolism of the assessed tissue. The study aimed to determine the reference values of rSO₂ for selected body areas, considering gender, age and body composition. We studied 70 healthy volunteers divided into two age groups (18–30 and >60 years). The rSO₂ was measured using NIRS in eighteen selected regions of interest (ROIs). Body composition analysis was carried out using dual-energy X-ray absorptiometry (DXA). Significant differences in rSO₂ values were found between almost all analyzed ROIs (p < 0.05) with a simultaneous lack of asymmetry between contralateral side of the body. The average rSO₂ values from the ROIs analyzed ranged from 40.34 ± 17.65% (Achilles tendon) to 69.94 ± 6.93% (tibialis anterior muscle). Age and the values of adiposity indices and the fat mass content are factors that may significantly reduce the rSO₂ value. In most ROIs, higher rSO₂ values were recorded for the younger group (p < 0.0001). The rSO₂ values at rest are area-specific in young and elderly healthy subjects. The changes in rSO₂, both in clinical assessment and research, should be interpreted taking into account the body area being assessed and individual factors such as age and body fat content.

1. Introduction

Near-infrared spectroscopy (NIRS) is a non-invasive method for monitoring the availability and use of oxygen by tissues, first introduced in 1977 by Jobsis [1]. It is an optical method based on the ease with which near-infrared light (NIR = 700 ± 1000 nm) passes through biological tissues (including bones, skin and muscles). The amount of light “recovered” after irradiation of the tissue by the sensor depends on the degree of radiation dispersion in the tissue and the degree of radiation absorption by chromophores. Only three molecules have been determined to influence NIR absorption during changes in tissue oxygen tension: deoxyhemoglobin (HHb)/oxyhemoglobin (HbO₂), myoglobin (HMb)/oxymyoglobin (O₂Mb) and cytochrome oxidase (cyt_ox). Hemoglobin and myoglobin contain an iron core within each heme, which varies its light absorption in the NIR range based on whether or not oxygen is bound to it. They absorb radiation in a precisely defined range of wavelength between 640 and 940 nm (the so-called “optical window”). Most biological tissues, as skin and bone, have relatively low light absorption property in this range, depending on water and lipids contess [2]. The NIRS method cannot differentiate between HbO₂ and O₂Mb or HHb and HMb, which means that NIRS signals are the result of the weighted average of oxygen saturations of the heme group of hemoglobin (Hb) in the vascular bed (small arteries, arterioles, capillaries, venules and small veins) and the heme group of myoglobin (Mb) in the muscle fibers [3]. Differences in the oxygen-dependent absorption spectra of iron and/or copper centers in these molecules enable measurement of relative changes in the amounts of oxidized copper and oxidized forms of heme present in muscles [4,5]. In NIRS, based on the absorption of near-infrared light by hemoglobin, as the absorption properties of hemoglobin change with its oxygenation status, measuring the transmitted/reflected light from tissue provides information on the oxygenation state of tissue [6]. The oxygen delivery (DO₂) to the tissues depends on the cardiac output (CO) and on the arterial O₂ content (CaO₂). CaO₂ describes the concentration of oxygen in arterial blood and represents the sum of oxygen bound to hemoglobin and oxygen dissolved in plasma, thus it is dependent on the hemoglobin (Hgb) concentration, the O₂ saturation (SaO₂) and the partial pressure of O₂ (PaO₂) [7].

This means that NIRS signals are the result of the weighted average of oxygen saturations of the heme group of hemoglobin (Hb) in the vascular bed (small arteries, arterioles, capillaries, venules and small veins) and the heme group of myoglobin (Mb) in the muscle fibers.

The NIRS method provides the ability to monitor the patient’s condition in real time and, above all, allows for the assessment and monitoring of tissue oxygenation and hemodynamics of tissue in vivo. Two different basic variables, tissue oxygen saturation (SPO₂) [8,9] and regional oxygen saturation (rSO₂), can be extracted, depending on the device used [10,11]. Regional oxygen saturation (rSO₂) is a measure of Oxy-Hb in a volume of tissue, expressed as a percentage of oxygenated hemoglobin to total hemoglobin (HbO₂/Hb) [12].

The rSO₂ value is believed to reflect the perfusion and metabolism of the tissue being assessed, assessing the balance of local tissue oxygen supply and demand. Tissue microcirculation includes arteries, veins and capillaries, which means that rSO₂ represents a “weighted average” of these structures, with approximately 75–85% of the signal coming from the venous vessels [13,14,15]. The first conducted research allowed for the determination of penetration depth at approximately 1/3 of the emitter–detector distance [16]. It was further assumed that when setting the detector–source system at a distance of 3 cm, the area of maximum sensitivity would be located at 1.5 cm deep below the skin layer [17].

The NIRS method has been widely adopted over the past two decades for research and is now increasingly being deployed for clinical use. It has been widely used in assessment of cerebral oxygenation in neonates [18,19] and adult patients [18]. NIRS is also becoming a widely used research instrument in the field of cognitive neuroscience [19].

There is also a constantly growing interest among researchers in the possibility of using NIRS in monitoring changes in muscle oxygenation and blood flow during physiological and pathological conditions, e.g., during submaximal and maximum exercise [13,20,21,22], to identify peripheral vascular disease [23], in chronic obstructive pulmonary disease (COPD) [24], in cardiovascular diseases [25] and in sepsis [26]. It has been found that during work the degree of deoxidation of skeletal muscles varies depending on the type of muscle, type of exercise and blood flow specific to a given area and health status [27,28]. Only a few authors refer to the use of NIRS in physiotherapy as a method for assessing the impact of physical or kinesiotherapy treatment or entire rehabilitation programs in therapeutic rehabilitation [3,29,30].

However, the lack of standard measurement procedures and reference data for region of interest (ROI) assessment is the main limitation of this technique, which is often highlighted by researchers [12]. It has not yet been assessed whether different body areas are characterized by the same or different values of rSO₂ at rest. Also, there is no established measurement site, which can lead to different values for variables when different muscles are used. Significant regional variations in rSO₂ values seem very likely to be due to natural differences in the morphological characteristics of each individual tissue (thickness, structure and optical properties through which NIR radiation potentially must penetrate), as well as microcirculatory properties, functions and metabolic states.

Understanding the differences in regional oxygen saturation in healthy young people under physiological conditions is crucial for the subsequent assessment of changes in saturation in different areas, both in detecting abnormal/pathological situations as well as in assessing the progress of treatment and rehabilitation in relation to a specific area of the body. Many therapeutic effects of rehabilitation are based on the stimulation of microcirculation and tissue metabolism, which means trophic effects in tissue regeneration. Currently, especially in physiotherapy practice, the assessment of this effect is often based on the patient’s subjective assessment.

It seems that NIRS will allow for an objective assessment of therapeutic effects in this aspect, but for these purposes it is necessary to know the specific physiological differences in regional saturation in healthy people. Therefore, the main goal of this pilot study was to determine reference values of regional oxygen saturation (rSO₂) for 18 selected body areas under physiological conditions in healthy young people aged 18–30 and to determine possible variations in their values depending on the body area, gender and age (compared to elderly people over 60 years of age). Additionally, the symmetry of rSO₂ values in opposite areas of the body in individual subjects was also assessed.

2. Materials and Methods

The research was approved by the Bioethics Committee of the Pomeranian Medical University in Szczecin (no. KB-006/66/2023). The research was carried out in the Diagnostic Laboratory of the Department of Functional Diagnostics and Physical Medicine of the Pomeranian Medical University in Szczecin in the period from June to September 2023.

2.1. Volunteers

Healthy people aged 18–30 (young group) and over 60 years (older group) were recruited for the study. The exclusion criteria were: unstabilized hypertension, hemoptysis, pneumothorax, recent myocardial infarction (up to 3 months ago), severe heart failure, respiratory arrhythmias, chronic obstructive pulmonary disease, asthma, unstable angina, diagnosed acute or critical limb ischemia, Buerger’s and Leriche’s syndrome, malignant neoplasm, anemia, chronic kidney disease as defined by creatinine levels above 2.5 mg/dL, retinal detachment/ophthalmological surgery within the last 6 months, recent stroke (up to 3 months ago), recent surgery on the head, chest or lower abdomen (up to 3 months ago), smoking/electronic cigarettes, receiving immunosuppressants while undergoing chemotherapy or radiotherapy, taking anticoagulants. Additionally, people with contraindications to dual-energy X-ray absorptiometry (DXA) were excluded [31].

A total of 70 participants have been included in the study: 30 young people aged 18–30 as a reference group and 40 elderly people over 60 years old. Before the research, all qualified participants were informed about the purpose of the project and gave their informed, written consent to participate in the research (in accordance with the Declaration of Helsinki). The course and sequence of the research procedure are presented schematically in Figure 1.

2.2. Research Procedures

The research was conducted in two stages. In the first stage, everyone underwent pre-qualification procedures to exclude possible peripheral circulation disorders and respiratory diseases. Then, participants without contraindications were qualified for the main research.

2.2.1. Pre-Qualification Tests

To exclude circulatory disorders, the ABI was measured. The ABI was performed by measuring the systolic blood pressure from brachial arteries and the dorsalis pedis and posterior tibial arteries after the patient had been at rest in the supine position for 10 min to normalize the blood pressure and heart rate. The measurement was performed using an AMPI MD (MESI) automated wireless ankle–brachial index system, automatically calculating the ankle–brachial index values. Acceptable ABI ranges were from 0.9 to 1.4 [32].

In order to exclude people with ventilation disorders, a slow spirometry test was performed. The examination was carried out in accordance with applicable standards for lung volume measurement [33] and the manufacturer’s recommendations. The measurement was performed in patients at the same time of day (morning, 8–10 a.m.), in a room with a humidity of 50% and a temperature of 23 ± 1 °C. Before performing the measurement, the volume was calibrated in accordance with the manufacturer’s recommendations. During the examination, the patient sat upright, with his shoulders slightly back, chin slightly raised, and a clip placed on the lower half of the nose. The patient was asked to lightly bite down on the mouthpiece. The actual measurement was preceded by a thorough explanation and demonstration of the procedure and test measurement. Each volunteer had three measurements, and their average values were used for statistical analyses. The test was performed on a Vyntus-Spiro diagnostic device (CareFusion Germany, Hoechberg, Germany) with SentrySuite™ software v.3.0 (Vyaire, Mettawa, IL, USA). The results obtained from the volunteers were each time compared to the norms, taking into account gender and age.

2.2.2. Regional Oxygen Saturation (NIRS) and Body Composition (DXA) Measurements

In the second stage of the study, volunteers who were qualified for the study based on pre-qualification tests had their saturation measured using the NIRS method and body composition analysis using DXA methods.

2.3. Regional Oxygen Saturation (rSO₂) Assessment

For this purpose, the SenSmart™ Model X-100 device (Nonin Medical, Inc., Playmouth, MN, USA) with SenSmart software v.1.0 (Nonin Medical, Inc.) was used. The device is based on the modified Beer–Lambert law. Peripheral oxygen saturation (SPO₂) was measured by placing a sensor on the index finger. The study used the 8000SM model, which uses the red wavelength = 660 nm and the infrared wavelength = 910 nm. The sensor was placed on the patient’s finger for the entire duration of the study. To measure regional oxygen saturation (rSO₂), an EQUANOX Advance Model 8004CA sensor was used. The sensor consists of 2 light-emitting diodes (located externally), each generating 4 near-infrared spectroscopy wavelengths (730, 760, 810 and 880 nm). There are 2 detection sensors between the emitters, each optode is 20 mm from the next. Each detector receives light from two emitters. The light that reaches the emitter from the closer detector propagates through the shallower part of the analyzed structure, while the light from the emitter to the further detector passes through the deeper structure. According to the manufacturer’s information, the sensor measurement depth is ~20 mm. The value of rSO₂ is given after a calculation using an unknown algorithm based on the two signals from the two sets of emitters and detectors. The stable sensor was attached to the skin over a fragment of tissue monitored symmetrically on the right and left side of the body to perform measurements without changing its position for 30 s. The device refreshes rSO₂ values every 1.8 s; the analyses were based on the average values obtained over the entire period of sensor use. Eighteen selected regions of interest (ROIs) on the anterior and posterior body sides are presented graphically in Figure 2.

2.4. Body Composition Analysis

Body composition analysis using DXA using a Hologic Horizon DXA System^® densitometer (Quirugil, Bogota, Colombia) with Discovery software, version 12.3 (Bellingham, WA, USA) was performed. In order to ensure the repeatability and reliability of the results, the device was calibrated every day of the study in accordance with the manufacturer’s recommendations. The examination and subsequent analysis of the scans were performed by one researcher in order to limit measurement error.

2.5. Statistical Analysis

The statistical analysis of the results was performed using Statistica 13.3 software (Statistica PL, StatSoft, Kraków, Poland). The Shapiro–Wilk test was used to analyze the normality of distribution. Normally distributed data were presented as mean values and standard deviation (SD). Values whose distribution deviated from normal were presented as median and minimum and maximum values (min–max). The significance of differences between independent variables with a normal distribution was analyzed using the Student’s t-test. A similar analysis for non-normally distributed variables was performed using the Mann–Whitney U test. The level of significance was set to p < 0.05. The relationship between the studied variables was examined using the Pearson correlation coefficient. In addition, in order to assess inequalities in the distribution of rSO₂ values in the different regions evaluated, the Gini index was calculated from the average for the right and left sides of the body.

3. Results

Characteristics of the Study Group

The final study group consisted of 70 healthy volunteers, of which 30 were a young group of volunteers with a median age of 23 (20–30) years and 40 were an older group of volunteers with a median age of 70 (60–79) years. Analysis of body composition parameters showed significantly higher median values of body mass index (BMI), percentage of body fat (%PBF) and fat mass (FM) (p = 0.01) in the older group compared to the younger group. With regard to tissue oxygen saturation parameters, the median SPO₂ value was 97.5% (92–100%) in the whole group and was significantly higher for the young participants compared to the older ones (p = 0.01). The median HR value for the entire group was 74 and, similarly, it was slightly higher for the younger group compared to the older group (p = 0.62). No intergroup differences were found in the ABI (p > 0.05). All of the analyzed spirometric parameters, except VT (VCmax, VCin, VCex, IC, IRV, ERV), were characterized by higher values in the young group compared to the older group (p = 0.01). The detailed values of the analyzed parameters, including the division into younger and older groups, are shown in

Table 1. Characteristics of the study group including anthropometric, body composition, hemodynamic and spirometric characteristics, including the division into a younger and an older group.

		Study Group n = 70 (♀ = 51, ♂ = 19)		Young Group n = 30 (♀ = 18, ♂ = 12)		Older Group n = 40 (♀ = 33, ♂ = 7)		Mann–Whitney U Test
		Median	Min–Max	Median	Min–Max	Median	Min–Max	z	p
Age [years]		64	20–79	23	20–30	70	60–79	−7.16	0.000
Weight [kg]		71	46–115	66.50	46–115	72.5	49–94	−1.61	0.079
Height [cm]		165.00	150.5–196	169.25	163–196	163	150.5–185	4.38	0.000
BMI [kg/m2]		26.24	16.2–38.7	22.42	16.21–38.88	27.74	20.13–37.8	−4.38	0.000
FM [kg]		26.29	10.20–55.85	20.50	10.21–53.93	28.14	12.76–55.85	−3.94	0.000
PBF [%]		36.3	17.02–49.33	31.48	17.03–49.34	39.79	20.77–47.17	−4.12	0.000
WBSAT [kg]		1.40	0.24–3.45	0.93	0.24–3.08	1.52	0.66–3.45	−4.30	0.000
SPO2		97.5	92–100	98	94–100	97	92–99	3.79	0.000
HR		74.5	53–116	74.5	53–100	74	57–116	−0.49	0.622
ABI	L	1.17	0.9–1.36	1.17	1.07–1.31	1.16	0.9–1.36	−0.15	0.884
	P	1.19	0.9–1.39	1.19	1.05–1.39	1.18	0.9–1.36	0.51	0.610
VCmax		3.52	1.6–7.31	4.45	2.57–7.31	3.03	1.6–5.18	5.25	0.000
VCin		3.52	1.38–7.31	4.45	1.91–7.31	2.99	1.38–4.97	4.82	0.000
VCex		2.77	0.77–7.01	3.72	1.51–7.01	2.09	0.77–5.18	4.45	0.000
IC		2.59	1.23–8.91	3.17	1.42–5.54	2.44	1.23–8.92	3.57	0.000
IRV		1.67	0.2–4.22	2.07	0.25–4.22	1.39	0.2–2.72	3.50	0.000
VT		1.00	0.4–8.04	1.02	0.4–3.08	0.93	0.44–8.04	1.12	0.263
ERV		0.88	0.11–6.98	1.33	0.33–2.39	0.65	0.11–6.98	4.82	0.000

Legend: BMI—body mass index; FM—fat mass; PBF—percentage of body fat; WBSAT—subcutaneous fat tissue; SPO₂—oxygen saturation; HR—heart rate; ABI—ankle–brachial index; Vc_max—maximum vital capacity; VC_in—inspiratory vital capacity; VC_ex—expiratory vital capacity; IC—inspiratory capacity; IRV—inspiratory reserve volume; VT—tidal volume; ERV—exhalation reserve volume.

.

A comparison of the rSO₂ values read from the corresponding ROIs on the right and left sides showed no significant asymmetry in all analyzed ROIs, except for the older group’s carpal tunnel area (p < 0.01) (

Table 2. A comparison of the rSO₂ values read from the corresponding ROIs on the right and left sides.

			Young Group n = 30		t-Test		Older Group n = 40		t-Test
			Mean [%]	±SD	t	p	Mean [%]	±SD	t	p
Head	Forehead	L	70.67	4.50	−0.23	0.819	64.73	7.31	−1.28	0.206
		R	70.97	5.54			66.70	6.52
Neck	Middle neck	L	73.03	6.00	0.40	0.689	67.80	5.74	0.10	0.924
		R	72.47	4.88			67.68	5.89
Trunk	Trapezius	L	71.97	4.25	−0.18	0.860	67.30	7.94	−0.03	0.975
		R	72.20	5.86			67.35	6.25
	C-Th	L	66.60	7.38	−0.15	0.882	61.75	7.93	0.98	0.330
		R	72.20	6.46			60.03	7.82
	Th-L	L	69.37	7.23	−0.34	0.737	62.63	7.23	−0.20	0.843
		R	69.97	6.55			62.95	7.41
	L-S	L	67.90	7.58	0.40	0.694	64.65	5.14	−0.74	0.459
		R	67.07	8.69			65.50	5.08
Upper limb	Wrist extensors	L	66.87	7.85	−0.31	0.757	65.43	7.26	−0.14	0.888
		R	67.57	9.52			65.65	6.97
	Cubital fossa	L	67.07	7.24	−0.04	0.970	64.53	8.30	−1.47	0.146
		R	67.13	6.20			66.95	6.34
	Carpal tunnel	L	52.93	11.93	−0.11	0.910	61.68	10.44	2.60	0.011
		R	53.27	10.76			56.33	7.78
Lower limb	Femoral triangle	L	69.00	9.24	0.30	0.769	60.70	8.83	−0.10	0.921
		R	68.27	9.99			60.90	9.08
	Rectus femoris	L	66.50	9.86	0.47	0.639	61.75	8.22	−0.19	0.847
		R	65.37	8.74			62.10	7.91
	Tibialis anterior	L	70.13	8.96	0.21	0.835	69.53	5.51	−0.76	0.447
		R	69.57	11.82			70.48	5.59
	Ankle joint	L	43.80	14.37	−0.71	0.482	52.69	10.58	−0.80	0.429
		R	46.62	16.20			54.59	10.49
	Biceps femoris	L	64.03	6.61	0.25	0.807	63.75	8.55	−0.24	0.807
		R	63.57	8.06			64.20	7.89
	Popliteal fossa	L	62.83	10.00	−0.23	0.818	59.65	8.29	0.10	0.917
		R	63.47	11.14			59.45	8.88
	Gastrocnemius	L	65.63	9.41	0.68	0.499	64.65	7.91	−0.33	0.743
		R	63.57	13.73			65.25	8.42
	Achilles tendon	L	27.40	18.46	−0.84	0.403	51.43	14.46	1.84	0.070
		R	31.43	18.63			45.65	13.64
	Plantar fascia	L	56.23	11.30	−0.20	0.842	61.58	12.04	−0.20	0.845
		R	56.80	10.57			62.10	11.93

). For this reason, the mean rSO₂ value calculated from the ROI on the right and left sides of the body was taken into account in analyses.

The mean rSO₂ values estimated in the entire study group and divided into young and older groups for selected areas of the body separately are presented in

Table 3. Mean values of rSO₂ obtained from the analyzed ROIs for the entire study group and the younger and older groups, including the analysis of intergroup differences.

		Study Group n = 70		Young Group n = 30		Older Group n = 40		t-Test
		Mean [%]	±SD	Mean [%]	±SD	Mean [%]	±SD	t	p
Head	Forehead	67.72	6.37	67.72	6.37	65.40	6.68	3.86	0.000
	Gini index	0.081		0.101		0.107
Neck	Middle neck	69.89	6.05	69.89	6.05	67.74	5.76	3.74	0.000
	Gini index	0.076		0.105		0.096
Trunk	Trapezius	69.36	6.35	69.36	6.35	67.33	6.71	3.32	0.001
	Gini index	0.078		0.102		0.104
	C-Th	63.39	7.73	63.39	7.73	60.89	7.63	3.36	0.001
	Gini index	0.097		0.122		0.119
	Th-L	65.74	7.76	65.74	7.76	62.79	7.20	4.06	0.000
	Gini index	0.097		0.117		0.110
	L-S	66.11	6.43	66.11	6.43	65.08	4.87	1.57	0.122
	Gini index	0.087		0.131		0.091
Upper limb	Wrist extensors	66.26	7.46	66.26	7.46	65.54	6.72	0.93	0.355
	Gini index	0.09		0.135		0.104
	Cubital fossa	66.32	6.28	66.32	6.28	65.74	6.46	0.90	0.373
	Gini index	0.082		0.116		0.105
	Carpal tunnel	56.47	9.69	56.47	9.69	59.00	8.37	−2.63	0.011
		0.125		0.177		0.128
Lower limb	Femoral triangle	64.16	9.74	64.16	9.74	60.80	8.84	3.61	0.001
	Gini index	0.114		0.14		0.130
	Rectus femoris	63.64	8.62	63.64	8.62	61.93	7.89	1.96	0.054
	Gini index	0.103		0.139		0.120
	Tibialis anterior	69.94	6.93	69.94	6.93	70.00	5.26	−0.09	0.929
	Gini index	0.081		0.131		0.091
	Ankle joint	49.80	12.76	49.80	12.76	53.64	9.25	−3.02	0.004
	Gini index	0.168		0.244		0.146
	Biceps femoris	63.90	7.54	63.90	7.54	63.98	8.00	−0.10	0.924
	Gini index	0.093		0.128		0.116
	Popliteal fossa	61.09	9.12	61.09	9.12	59.55	7.85	1.66	0.102
	Gini index	0.108		0.152		0.120
	Gastrocnemius	64.80	8.60	64.80	8.60	64.95	7.80	−0.17	0.868
	Gini index	0.102		0.148		0.117
	Achilles tendon	40.34	17.65	40.34	17.65	48.54	13.33	−5.29	0.000
	Gini index	0.275		0.388		0.201
	Plantar fascia	59.71	11.90	67.72	6.37	61.99	12.15	−1.81	0.075
	Gini index	0.139		0.173		0.156

.

Intergroup comparative assessment showed the occurrence of significantly higher rSO₂ values in the younger group in the following areas: forehead (p < 0.0001), middle neck (p < 0.0001), trapezius (p = 0.001), C-TH (p < 0.0001), TH-L (p < 0.0001), L-S (p = 0.036), femoral triangle (p = 0.001) (Table 3). Lower values of the rSO₂ index in the younger group compared to the older group were found in the areas of carpal tunnel (p = 0.011), ankle joint (p = 0.004) and Achilles tendon (p < 0.0001). However, there were no significant differences between the younger and older groups in the following areas: trapezius L-S, wrist extensors, cubital fossa, rectus femoris, tibialis anterior, gastrocnemius, popliteal fossa and plantar fascia (p > 0.05).

The results of the analysis of gender differences in rSO₂ values from the assessed ROIs are presented in

Table 4. Gender differences in the study groups.

		Women n = 51		Men n = 19		t-Test
		Mean [%]	±SD	Mean [%]	±SD	t	p
Head	Forehead	66.75	6.48	70.33	5.39	−2.15	0.035
Neck	Middle neck	68.76	5.98	72.89	5.30	−2.65	0.010
Trunk	Trapezius	68.91	6.40	70.58	6.20	−0.98	0.332
	C-Th	62.77	8.29	65.05	5.86	−1.10	0.276
	Th-L	65.09	8.33	67.47	5.80	−1.15	0.256
	L-S	65.31	5.99	68.24	7.22	−1.72	0.091
Upper limb	Wrist extensors	65.15	7.63	69.24	6.20	−2.09	0.040
	Cubital fossa	66.22	6.38	66.61	6.16	−0.23	0.819
	Carpal tunnel	55.54	9.93	58.97	8.81	−1.33	0.190
Lower limb	Femoral triangle	63.25	9.95	66.61	8.95	−1.29	0.202
	Rectus femoris	61.55	7.72	69.26	8.59	−3.61	0.001
	Tibialis anterior	69.56	6.42	70.95	8.25	−0.75	0.460
	Ankle joint	50.92	12.53	46.84	13.25	1.19	0.239
	Biceps femoris	62.85	7.17	66.71	7.98	−1.95	0.056
	Popliteal fossa	59.38	9.27	65.68	7.04	−2.69	0.009
	Gastrocnemius	64.10	8.23	66.68	9.50	−1.13	0.266
	Achilles tendon	44.16	15.56	30.11	19.23	3.15	0.002
	Plantar fascia	60.21	11.74	58.39	12.53	0.57	0.575

. In the group of women, significantly lower rSO₂ values were found in the forehead (p = 0.035), middle neck (p = 0.01), wrist extensors (p = 0.04) and rectus femoris (p = 0.001) as well as a popliteal fossa (p = 0.009) and higher rSO₂ values in the Achilles tendon (p = 0.002) have been obtained compared to men. However, due to the large differences in numbers between the groups of women and men, these results should be treated cautiously. In the presented study, the group of men was too small for detailed cross-gender analyses within the young and old groups.

In the next stage of the analysis, interarea differences in rSO₂ values were assessed. It was observed that almost all assessed ROIs differed significantly in mean rSO₂ values, both in the entire study group and in the younger and older groups analyzed separately.

The detailed results of the interarea analysis are presented in

Table 5. Differences between ROIs for the entire group (Y + O) and young (Y) and old (O) groups separately.

ROI			Neck	Trunk				Upper Limb			Lower Limb
			Middle Neck	Trapezius	C-Th	Th-L	L-S	Wrist Extensors	Cubital Fossa	Carpal Tunnel	Femoral Triangle	Rectus Femoris	Tibialis Anterior	Ankle Joint	Biceps Femoris	Popliteal Fossa	Gastrocnemius	Achilles Tendon	Plantar Fascia
Head	Forehead	Y + O	−2.59 *	−1.83	5.13 ***	2.49 *	2.25 *	1.89	1.91	10.74 ***	3.70 ***	4.57 ***	−2.32 *	14.13 ***	4.61 ***	6.91 ***	3.10 *	16.39 ***	7.31 ***
		Y	−2.03 *	−1.38	3.72 ***	1.05	2.71 **	2.79**	3.45 **	11.14 ***	1.57	3.59 ***	0.65	12.38 ***	6.13 ***	5.11 ***	3.78 ***	16.73 ***	9.27 ***
		O	−2.00 *	−1.45	4.11 ***	2.60 *	0.66	0.16	−0.02	5.09 ***	3.89 ***	3.19 **	−4.31 ***	8.53 ***	1.45	5.01 ***	0.64	9.68 ***	2.51 *
Neck	Middle neck	Y + O	x	0.68	7.63 ***	4.93 ***	4.95 ***	4.32***	4.48 ***	12.88 ***	5.80 ***	6.90 ***	−0.06	15.95 ***	7.14 ***	9.16 ***	5.21 ***	17.73 ***	9.23 ***
		Y	x	0.69	5.32 ***	2.73 **	4.19 ***	4.19***	5.08 ***	12.18 ***	2.90 **	4.92 ***	1.91	13.20 ***	7.61 ***	6.29 ***	4.89 ***	17.41 ***	10.36 ***
		O	x	0.4	6.27 ***	4.77 ***	3.09 **	2.15*	1.9	7.01 ***	5.85 ***	5.26 ***	−2.53 *	10.48 ***	3.36 **	7.10 ***	2.50 **	11.16 ***	3.99 ***
Trunk	Trapezius	Y + O		x	6.77 ***	4.15 ***	4.08 ***	3.57***	3.67 ***	12.09 ***	5.13 ***	6.13 ***	−0.65	15.27 ***	6.28 ***	8.37 ***	4.55 ***	17.25 ***	8.58 ***
		Y		x	4.85 ***	2.20 *	3.73 ***	3.76***	4.60 ***	11.90 ***	2.47 *	4.51 ***	1.49	12.97 ***	7.21 ***	5.93 ***	4.54 ***	17.23 ***	10.06 ***
		O		x	5.43 ***	3.99 ***	2.30 *	1.6	1.38	6.26 ***	5.13 ***	4.51 ***	−2.66 **	9.61 ***	2.77 **	6.26 ***	1.97	10.54 ***	3.54 **
	C-Th	Y + O			x	−2.48 *	−3.09 **	3.04**	3.24 **	−6.14 ***	−0.71	−0.25	−6.87 ***	10.14 ***	−0.54	2.18 *	−1.32	13.39 ***	3.20 **
		Y			x	−2.34 *	−0.55	0.34	0.3	−8.00 ***	−1.25	0.54	−1.94	9.97 ***	2.26 *	2.21 *	1.22	14.65 ***	6.16 ***
		O			x	−1.59	−3.99 ***	3.93***	4.01 ***	−1.37	0.07	−0.83	−8.47 ***	4.91 ***	−2.43 *	1.03	−3.21 **	6.78 ***	−0.6
	Th-L	Y + O				x	−0.43	0.56	0.65	−8.27 ***	1.47	2.10 *	−4.44 ***	11.97 ***	1.96	4.44 ***	0.88	14.79***	5.18***
		Y				x	1.6	−1.72	−2.08 *	−9.74 ***	0.68	2.51 *	−0.11	11.34 ***	4.54***	4.03 *	2.89**	15.81 ***	7.94 ***
		O				x	−2.30 *	2.42*	2.54 **	−2.82 **	1.55	0.71	−7.05 ***	6.37 ***	−0.97	2.58 **	−1.77	7.96 ***	0.61
	L-S	Y + O					x	0.17	0.26	−9.07 ***	1.93	2.65 **	−4.37 ***	12.73 ***	2.55 *	5.10 ***	1.31	15.34 ***	5.75 ***
		Y					x	−0.17	−0.28	−8.03 ***	−0.71	0.98	−1.39	10.00 ***	2.61 **	2.53 ***	1.57	14.60 ***	6.27 ***
		O					x	0.47	0.66	−5.03 ***	3.73 ***	2.97 **	−5.85 ***	8.73 ***	1.02	4.97 ***	0.12	9.76 ***	2.24 *
Upper limb	Wrist extensors	Y + O						x	−0.07	8.75 ***	1.97	2.63 **	−3.90 ***	12.38 ***	2.53 *	4.95 ***	1.38	15.11 ***	5.63 ***
		Y						x	0.08	7.70 ***	−0.85	0.78	−1.51	9.72 ***	2.34 *	2.31 *	1.39	14.34 ***	5.97 ***
		O						x	−0.17	4.93 ***	3.73 ***	3.02 **	−4.44 ***	8.37 ***	1.29	4.83 ***	0.49	9.55 ***	2.39 *
	Cubital fossa	Y + O							x	9.07 ***	2.09	2.80 **	−4.00 ***	12.70 ***	2.71 **	5.19 ***	1.49	15.33 ***	5.83 ***
		Y							x	8.28 ***	−1.02	0.79	−1.72	10.19 ***	2.58 *	2.46 *	1.44	14.84 ***	6.43 ***
		O							x	4.98 ***	3.81 ***	3.12 **	−4.11 ***	8.37 ***	1.43	4.89 ***	0.64	9.56 ***	2.48 **
	Carpal tunnel	Y + O								x	−6.23 ***	−6.14 ***	−11.96 ***	4.45 ***	−6.65 ***	−3.81 ***	−6.80 ***	8.85 ***	−2.30 *
		Y								x	−8.15 ***	−6.82 ***	−8.46 ***	3.23 ***	−6.17 ***	−5.06 ***	−5.48 ***	8.47 ***	−1.69
		O								x	−1.23	−2.10 *	−8.92 ***	3.36 **	−3.54 **	−0.38	−4.25 ***	5.45 ***	−1.66
Lower limb	Femoral triangle	Y + O									x	0.46	−5.37 ***	10.01 ***	0.24	2.63 **	−0.55	13.28 ***	3.54 ***
		Y									x	1.57	−0.67	10.08 ***	3.11 **	2.99 **	2.07 *	14.59 ***	6.49 ***
		O									x	−0.84	−7.85 ***	4.62 ***	−2.35 *	0.91	−3.08 **	6.52 ***	−0.62
	Rectus femoris	Y + O										x	−6.29 ***	10.06 ***	−0.26	2.33 *	−1.04	13.33 ***	3.30 **
		Y										x	−2.18 *	9.00 ***	1.4	1.54	0.69	13.67 ***	5.11 ***
		O										x	−7.41 ***	5.58 ***	−1.6	1.81	−2.37 *	7.32 ***	0.05
	Tibialis anterior	Y + O											x	15.11 ***	6.41 ***	8.41 ***	4.82 ***	17.20 ***	8.66 ***
		Y											x	10.33 ***	3.69 ***	3.51 **	2.60 *	14.76 ***	6.87 ***
		O											x	12.28 ***	5.46 ***	9.18 ***	4.59 ***	12.54 ***	5.56 ***
	Ankle joint	Y + O												x	−10.59 ***	−7.95 ***	−10.52 ***	4.93 ***	−6.32 ***
		Y												x	−8.52 ***	−7.50 ***	−7.80 ***	5.07 ***	−4.68 ***
		O												x	−6.91 ***	−3.88 ***	−7.58 ***	2.55 **	−4.58 ***
	Biceps femoris	Y + O													x	2.69 **	−0.85	13.74 ***	3.65 ***
		Y													x	0.39	−0.45	13.39 ***	4.31 ***
		O													x	3.35 **	−0.76	8.40 ***	1.32
	Popliteal fossa	Y + O														x	−3.21 **	11.67 ***	1.2
		Y														x	−0.71	12.28 ***	3.40 **
		O														x	−4.10 ***	5.92 ***	−1.4
	Gastrocnemius	Y + O															x	13.69 ***	4.06 ***
		Y															x	12.45 ***	3.92 ***
		O															x	8.94 ***	1.93
	Achilles tendon	Y + O																x	−10.28 ***
		Y																x	−9.79 ***
		O																x	−6.40 ***

Legend: * p < 0.05, ** p < 0.01, *** p < 0.001.

for the entire group and the younger and older groups, respectively. The mean rSO₂ value in the forehead was 67.90 ± 6.67 and was significantly lower only in comparison to the middle neck and tibialis anterior. Simultaneously, the mean value of rSO₂ in the forehead was significantly higher compared to all analyzed ROIs on the back (C-Th, Th-L, L-S) and in the limbs (carpal tunnel, femoral triangle, rectus femoris, ankle joint, biceps femoris, popliteal fossa gastrocnemius, Achilles tendon, plantar fascia) (p < 0.05) (Table 5). Similar results were obtained when comparing the forehead to other ROIs performed with a division into the younger and older groups. In both the younger and older groups, the rSO₂ value in the forehead area is lower than in the middle neck and tibialis anterior but higher for the following ROIs: C-Th, carpal tunnel, rectus femoris, ankle joint, popliteal fossa, Achilles tendon and plantar fascia. Higher values for the forehead were also confirmed for the ROIs Th-L and femoral triangle in the older group and for the ROIs L-S, wrist extensors, cubital fossa, biceps femoris and gastrocnemius in the younger group.

The mean value of the middle neck was 69.89 ± 6.14. It was shown to be higher in relation to almost all analyzed ROIs (i.e., C-Th, Th-L, carpal tunnel, femoral triangle, rectus femoris, ankle joint, biceps femoris, popliteal fossa, gastrocnemius, Achilles tendon, plantar fascia) both in the entire study group and the younger and older groups analyzed separately. Significantly higher rSO₂ values for the middle neck were also found compared to the L-S, wrist extensors and cubital fossa in the entire study group and the young group. In relation to the older group, a significantly lower value in the middle neck compared to tibialis anterior was also found.

The mean value of the trapezius was 69.36 ± 6.72, being significantly higher for every group arrangement (entire study group, young and older group) compared to ROIs on the back (C-Th, Th-L, L-S), carpal tunnel on the upper limb and most ROIs on the lower limb (femoral triangle, rectus femoris, ankle joint, biceps femoris, popliteal fossa, Achilles tendon, plantar fascia). Moreover, higher rSO₂ values of the trapezius in the entire group and the younger group were demonstrated compared to the remaining ROIs: wrist extensors, cubital fossa and gastrocnemius. Only in the older group was the trapezius rSO₂ value higher than that of the tibialis anterior.

The mean rSO₂ value from the C-Th was 63.39 ± 7.98, being significantly higher compared to the ankle joint and Achilles tendon in every group arrangement. Both in the entire group and in the younger group, the rSO₂ value of the C-Th was significantly lower compared to the Th-L and carpal tunnel and higher compared to the popliteal fossa and plantar fascia. In relation to the entire study group and the older group, the rSO₂ value of the C-Th was lower than the L-S and tibialis anterior and higher than the wrist extensors and cubital fossa. Additionally, the rSO₂ of the C-Th was higher than that of the gastrocnemius, but only in the older group.

The mean rSO₂ value from Th-L was 65.74 ± 7.85, being significantly lower compared to the carpal tunnel and, at the same time, significantly higher compared to the following ROIs in all group analyses: ankle joint, popliteal fossa and Achilles tendon. In the entire group, the rSO₂ value from the Th-L was lower than that of the tibialis anterior and higher than that of the rectus femoris and plantar fascia. In the young group, the rSO₂ value from the same ROI was lower than that of the cubital fossa and higher than that of the rectus femoris, biceps femoris, gastrocnemius and plantar fascia. In the group with elderly people, the value of rSO₂ of the Th-L was significantly lower compared to L-S and tibialis anterior and higher compared to wrist extensors and cubital fossa.

The mean rSO₂ value in the L-S was 66.11 ± 6.64, which was both lower than that of the carpal tunnel and higher than that of the ankle, popliteal fossa, Achilles tendon and plantar fascia, both in the entire study group and divided into the younger and older groups. Additionally, in the entire group, the rSO₂ value from the L-S was significantly lower than that of the tibialis anterior and significantly higher than that of the rectus femoris and biceps femoris. In the younger group, the rSO₂ value from the L-S was significantly higher compared to the biceps femoris, while in the older group it was significantly higher than that of the femoral triangle, rectus femoris and tibialis anterior.

The mean rSO₂ values for the wrist extensors were 66.26 ± 7.81, being significantly higher compared to the carpal tunnel, ankle joint, popliteal fossa, Achilles tendon and plantar fascia in the entire study group as well as in the younger and older groups. rSO₂ values from the wrist extensors were significantly lower than that of the tibialis anterior and higher than that of the rectus femoris in the entire study group and the older group. Additionally, higher values of wrist extensors were demonstrated compared to femoral triangle in the older group and biceps femoris in the entire study group and the younger group.

The mean rSO₂ value from the cubital fossa was 66.32 ± 7.13, being significantly higher than the rSO₂ value from the carpal tunnel, ankle joint, popliteal fossa, Achilles tendon and plantar fascia in the entire study group and after division into the younger and older groups. Additionally, in the entire study group and in the older group, the rSO₂ value from the cubital fossa was significantly higher compared to the rectus femoris and lower than that of the tibialis anterior and significantly higher than the rSO₂ of the biceps femoris in the entire study group and the femoral triangle in the older group.

The mean rSO₂ value from the carpal tunnel was 56.47 ± 10.68, being significantly lower than that of the rectus femoris, tibialis anterior, biceps femoris and gastrocnemius and, at the same time, significantly higher than that of the ankle joint and Achilles tendon for the entire study group and after division into younger and older groups. Additionally, for the entire study group and the younger group, the rSO₂ value from the carpal tunnel was significantly lower than that from the femoral triangle popliteal fossa and only for the entire study group relative to the plantar fascia.

The mean rSO₂ value from the femoral triangle was 64.16 ± 9.94, being significantly higher compared to the ankle joint and Achilles tendon in the entire study group and after division into the younger and older groups. Additionally, for the entire study group, the rSO₂ value from the femoral triangle was significantly lower than the rSO₂ from the tibialis anterior and higher than that of the popliteal fossa and plantar fascia. For the younger group analyzed separately, the rSO₂ value from the femoral triangle was additionally significantly higher than that of the biceps femoris, popliteal fossa, gastrocnemius and plantar fascia. However, for the older group, the rSO₂ value from the femoral triangle was significantly lower than that of the tibialis anterior, biceps femoris and gastrocnemius.

The mean rSO₂ value from the rectus femoris was 63.64 ± 8.77, being significantly higher compared to the ankle joint and Achilles tendon and, at the same time, significantly lower than that of the tibialis anterior for the entire study group and after division into the younger and older groups. Additionally, the rSO₂ value from the rectus femoris was significantly higher for the entire study group than that of the popliteal fossa and plantar fascia. Additionally, the rSO₂ value from the rectus femoris was significantly higher than that of plantar fascia for the younger group and lower than that of gastrocnemius for the older group.

The mean rSO₂ value from the tibialis anterior was 69.94 ± 7.96, being significantly higher compared to all other ROIs determined in the lower limb (ankle joint, biceps femoris, popliteal fossa, gastrocnemius, Achilles tendon, plantar fascia), both in the entire study group and after dividing into the younger and older groups.

The mean rSO₂ value from the ankle joint was low, averaging 50 ± 13.38, being significantly lower than the rSO₂ value from the biceps femoris, popliteal fossa, gastrocnemius and plantar fascia, and higher only than that of the Achilles tendon.

The mean rSO₂ value from the biceps femoris was 63.98 ± 8.18 and was significantly higher than the values from the popliteal fossa, Achilles tendon and plantar fascia in the entire study group. For the separately analyzed younger group, the average rSO₂ value from the biceps femoris was significantly higher than the rSO₂ value from the Achilles tendon and plantar fascia. In the older group, rSO₂ from the biceps femoris was significantly higher than the rSO₂ value from the popliteal fossa and Achilles tendon.

The mean rSO₂ value from the popliteal fossa was 61.09 ± 9.56 and was significantly lower than the rSO₂ value from the gastrocnemius in the entire study group and in the older group and significantly higher compared to the rSO₂ value from the Achilles tendon for the entire study group and after division into the younger and older groups. Additionally, the rSO₂ value from the popliteal fossa was significantly higher than the rSO₂ value from the plantar fascia in the younger group.

The mean rSO₂ value from gastrocnemius was 64.8 ± 9.79, from plantar fascia 59.56 ± 11.73 and Achilles tendon only 40.34 ± 18.74. The mean value of rSO₂ from the gastrocnemius was higher than rSO₂ from the Achilles tendon for all groups (younger and older groups) and higher than rSO₂ from the plantar fascia in the entire tested group and the younger group. The rSO₂ value from the Achilles tendon was significantly lower than the rSO₂ from the plantar fascia in all analyzed groups.

A detailed analysis of the Gini index values revealed that the average rSO₂ values from individual regions, assessed in the entire study group as well as separately for the younger and older groups, exhibited a fairly even distribution/concentration of data (Gini index < 0.3). Only the mean rSO₂ values recorded from the Achilles tendon in the younger group had a higher Gini coefficient value (0.338), indicating a moderate distribution/concentration of the data collected in this area (Table 3).

The results of the correlation analysis of rSO₂ values from the analyzed ROIs with selected anthropometric characteristics and age are shown in

Table 6. Relationship between values of selected anthropometric characteristics and age and rSO₂ values.

rSO2 [%]		Age [Year]	BMI [kg/m2]	PBF [%]	WBSAT [kg]	FM [kg]	LM [kg]
						Head
Head	Forehead	−0.45 ***	−0.12	−0.10	−0.12	0.17	0.07
						Trunk
Trunk	Middle neck	−0.39 **	−0.44 ***	−0.38 **	−0.34 **	−0.32 **	0.09
	Trapezius	−0.37 **	−0.66 ***	−0.41 **	−0.39 *	−0.40 ***	−0.03
	C-Th	−0.36 **	−0.67 ***	−0.45 ***	−0.37 *	−0.36 *	−0.04
	Th-L	−0.45 ***	−0.70 ***	−0.51 ***	−0.47 ***	−0.50 ***	−0.13
	L-S	−0.19	−0.45 ***	−0.45 ***	−0.43 ***	−0.42 ***	−0.03
						Upper limb
Upper limb	Wrist extensors	−0.06	−0.49 ***	−0.33 **	−0.35	0.08 **	0.24
	Cubital fossa	−0.14	−0.22	−0.32 **	−0.21 *	−0.00	0.24
	Carpal tunnel	0.29	0.33 **	0.07	0.19	0.13	0.18
						Lower limb
Lower limb	Femoral triangle	−0.36 **	−0.56 ***	−0.44 ***	−0.43 ***	−0.31 *	0.17
	Rectus femoris	−0.20	−0.46 ***	−0.45 ***	−0.42 ***	−0.40 ***	0.22
	Tibialis anterior	−0.06	−0.45 ***	−0.40 **	−0.42 ***	−0.46 ***	−0.11
	Ankle joint	0.34 **	0.11	0.10	015	−0.12	−0.24
	Biceps femoris	0.04	−0.33 **	−0.29 *	−0.24	−0.38 **	0.12
	Popliteal fossa	−0.14	−0.46 ***	−0.47 ***	−0.47 ***	−0.53 ***	0.14
	Gastrocnemius	−0.01	−0.31 **	−0.50 ***	−0.28 *	−0.41 ***	0.13
	Achilles tendon	0.51 ***	0.34 **	0.35 **	0.30 *	0.01	−0.37 **
	Plantar fascia	0.22	−0.04	−0.07	−0.78	−0.21	−0.06

Legend: FM—fat mass; WBSAT—whole body subcutaneous fat tissue; LM—lean mass; * p < 0.05, ** p < 0.01, *** p < 0.001.

. There was a significant relationship between the decrease in rSO₂ value with the age of the subjects in the following ROIs: forehead, middle neck, trapezius, C-Th, Th-L and femoral triangle, and between an increase in the rSO₂ value from the ankle joint and Achilles tendon and the age of the subjects (p < 0.01; p < 0.001). The study showed a significant negative correlation between the values of adiposity indices (BMI, %PBF) and fat mass content (WBSAT, FM) and rSO₂ values for most of the analyzed ROIs (p < 0.05, p < 0.01, p < 0.001). Except for the negative correlation of the rSO₂ value with the Achilles tendon, no relationship was found between the rSO₂ value and the LBM content in the analyzed ROIs (p < 0.01).

4. Discussion

To our knowledge, this is the first study to assess the normative values of the regional oxygen saturation (rSO₂) in selected areas of the body, in healthy people, taking into account age and gender. For this purpose, 18 ROIs on the front and back surfaces of the body were determined independently. The research was conducted in two groups of healthy volunteers, young and old, in the age range from 20 to 30 years and from 60 to 79 years, respectively. The average values of the rSO₂ from selected ROIs from the anterior and posterior sides of the upper and lower parts of the body have been measured. The 18 ROIs have been chosen because of their frequent involvement in overload injuries and following physiotherapy and their easy accessibility to NIRS techniques. The analysis began with assessing the symmetry of the distribution of the obtained saturation values for contralateral body areas, and then the comparative assessment of the rSO₂ values for individual areas was carried out. All analyses were performed independently for the entire group of subjects and then they were divided into groups, taking into account age and gender. Taking into account previous reports [34] on the relationship between changes in hemodynamic parameters (related to a sudden change in the position from lying down to standing) and the tissue saturation values recorded, especially in the distal parts of the body, we conducted our research on volunteers resting for 20 min in a lying position. Due to the above, reference to the values presented by us should be made not only considering the exact location of the sensor but also the lying position.

Peripheral muscle rSO₂ is a hemodynamic variable that differs between healthy and severely ill patients [35] and is associated with clinically important outcomes [36]. Available studies were performed using different NIRS pulse oximeters and at different body sites, making standardization of measurements and establishing reference values much more difficult and this is a significant obstacle to widespread clinical use. A fundamental problem of NIRS tissue oximetry is the lack of a reference standard. Therefore, testing and comparing commercially available NIRS equipment at different locations and in different populations is important, as other researchers have also pointed out [37].

The median absolute resting rSO₂ values recorded in the area of the proximal part of the forearm flexor muscles by the three leading pulse oximeters (INVOS 5100C = 70.7%, FORE-SIGHT = 64.6%, NONIN EQUANOX 7600 = 68.4%) in the steady state were within 6 percentage points, while the values during deoxygenation varied significantly. Therefore, forearm rSO₂ values using INVOS, NONIN and FORE-SIGHT in occlusion testing cannot be used interchangeably. The available literature results do not determine which device is best for assessing rSO₂. Although the NONIN we used was characterized by the highest variability of measurements, at the same time, the results of comparisons of pulse oximeters suggest that good repeatability comes at the expense of low sensitivity to changes in oxidation [37].

Measurement of rSO₂ finds application in the detection of tissue hypoperfusion, which, being a pathophysiological process, leads to multiple organ dysfunction and death. In patients at risk of hypoperfusion, it is crucial to monitor and detect inadequate tissue perfusion and oxygenation early. In clinical practice, tissue oxygenation is assessed by global measurements such as blood pressure and cardiac output or with invasive methods measuring variables derived from oxygen and blood lactate levels. The use of NIRS appears to be an important and valuable alternative because it is non-invasive, allows real-time measurements and is clinically reliable [38].

It is important to note the lack of significant differences between rSO₂ values recorded from symmetric ROIs on the right and left sides of the body, demonstrated both by us (Table 2) and in the results of other authors in healthy volunteers [34] and patients with respiratory sepsis [12]. The above has an important clinical implication that rSO₂ measurements do not need to be performed bilaterally or on the dominant side. This is particularly important in the case of local factors contraindicating or interfering with rSO₂ measurements, such as local injections, tattoos, bandage applications or kinesiology taping.

Nowadays, the use of NIRS has expanded significantly beyond the assessment of cerebral oxygenation and is increasingly enabling the monitoring of local tissue and muscle oxygenation and perfusion [13,39]. This paper focuses on the use and utility of NIRS in monitoring tissue/muscle oxygenation, leaving aside the already well-developed aspect of assessing cerebral perfusion in the literature. However, the assessment of rSO₂ in the forehead area, as one of the most frequently analyzed areas in the first research on the use of NIRS, was not abandoned. Muscular activation and muscle oxygenation are considered to be the main approaches in the monitoring of working muscle. From this perspective, NIRS was used in our study as a measurement to establish reference values for regional oxygen saturation. The research results published so far mainly concern lower extremity muscles (i.e., biceps femoris, gastrocnemius, rectus femoris, tibialis anterior, vastus lateralis, vastus medialis) [40,41] and to a lesser extent the muscles of the upper limbs (i.e., biceps brachii, brachioradialis muscle, shoulder muscle, forearm flexors, triceps brachii) [41,42]. Only a few studies refer to the saturation of the trunk muscles (i.e., dorsal extensor, intercostal, multifidus, condylar and anterior dentate) [43]. Although many of the superficial skeletal muscles were assessed by independent authors, no regional oxygen saturation reference values have been proposed. Moreover, almost all of the evaluations have been carried out regarding the influence of various types of exercise (weight training, running, cycling, rowing, Wingate test) or physical stimulation (electrical stimulation, whole-body vibration) on muscle saturation [13,44,45,46]. We would like to point out that it is difficult to assess the exact impact of therapeutic treatment or physical activity without knowledge about the resting values for a given area, as well as the individual factors that influence the regional saturation values.

Knowledge of normative reference values for individual ROIs seems to be particularly useful in assessing the condition of a patient staying in an intensive care unit. Patients in a critical condition due to cardiovascular disorders and respiratory failure are also characterized by worse parameters of microcirculation and perfusion of tissues sensitive to hypoxia, e.g., muscles [38]. The correct rSO₂ value is an indicator of maintaining the balance between the supply and demand for tissue oxygen and may also be an important clinical criterion for the patient’s prognosis [12,34].

Rodríguez et al. determined that an rSO₂ value < 60% recorded from the brachioradialis muscle at admission to the ICU is associated with a significant increase in mortality in patients with sepsis. The authors concluded that the rSO₂ value may be a useful tool for quantitative assessment of microcirculatory dysfunction but also for predicting the development of critically ill patients [12], which has also been shown in previous studies [8,11,47].

Normative reference values for individual ROIs may also be useful in assessing the condition of a patient in the intensive care unit and the effectiveness of resuscitation in situations of sudden cardiac arrest. Studies have confirmed the possibility of monitoring skeletal muscle rSO₂ in hospital cardiac arrest. Patients whose spontaneous circulation was restored had higher skeletal muscle rSO₂ at the beginning of advanced life support (basal rSO₂) and during cardiopulmonary resuscitation (maximal rSO₂) [48].

In the presented study, we accurately determined ROIs for the most commonly assessed skeletal muscles of the upper and lower extremities and back, as well as additional ROIs for tendons (Achilles, plantar fascia) and joint areas (cubital fossa, popliteal fossa and carpal tunnel) (Figure 2). We have provided age- and gender-specific reference values) (Table 3 and Table 4). The mean rSO₂ value for the analyzed ROIs ranged from the lowest values for the Achilles tendon (40.34%) to the highest values for tibialis anterior (69.94%). This distribution of values was independent of age and gender. Significant differences in rSO₂ values were found between all of the selected ROIs (Table 5). Such differentiation is confirmed by literature data, which show higher rSO₂ values in deltoid muscle than brachioradialis muscle. This clearly indicates area differences in rSO₂ values related to the blood supply and metabolic activity of tissues. Our findings support the conclusion that NIRS-derived values are not universal for all areas and should be carefully interpreted according to the underlying physiology of the monitored tissue.

Such results are consistent with the assumption that human skeletal muscle is composed of a heterogeneous collection of muscle fiber types. Most skeletal muscles in a human contain all three types of fibers: type I (slow-twitch oxidative), IIa (fast oxidative glycolytic (FOG)) and IIx (fast glycolytic (FG)), although in varying proportions. Moreover, even amongst fibers of the same type, there are structural and functional characteristic differences [44,49,50]. In addition, given the importance of the capillary bed for the delivery of oxygen to the muscle, it is expected that highly oxidative muscles have a denser capillary network than highly glycolytic muscles [51].

We also noticed that higher rSO₂ values were recorded for muscle ROIs (from 63.39% of the C-Th to 69.89% of the mid-neck) than for tendon and joint areas (from 40.34% of the Achilles tendon to 66.32% of the ulnar fossa) (Table 3). This seems to confirm once again the need to standardize the measurement of the signal site, as other researchers have also stressed [12]. However, only a few studies have tried to quantify the oxygen consumption of human tendon in vivo. The tendon’s blood circulation is poor, mostly peripheral (peritendinous sheet), and rises from the muscular belly and periosteum, although tendons are metabolically active tissues [52].

In Calanni et al.’s research, the usefulness of NIRS to study oxygen extraction and blood flow in the patellar tendon during ramped isometric knee extensions in healthy young subjects has been confirmed [53]. Similarly, Kubo et al., using NIRS and red laser light, found that the oxygen consumption of the Achilles tendon as well as that of the medial gastrocnemius muscle increased significantly after repeated muscle contractions [54].

This result implied that tendons as well as muscles consumed oxygen during contraction and changes in oxygen consumption within the tendon need to be elucidated to understand human tendon physiology in vivo.

The regional oxygen saturation index can also be used to evaluate the effect of therapeutic, rehabilitative interventions and new therapies on the status and possible changes in tissue perfusion. For example, rSO₂ was used to assess the effect of a lower limb elevation procedure after injury or surgery on the perfusion status of the tissues. The analysis of rSO₂ values at various degrees of elevation of the control and experimental limbs showed that as the elevation of the human limb increases, muscle oxygen saturation deteriorates, which may have important clinical implications [55]. Another study assessed whether reperfusion slope-based NIRS would detect the effects of a 12-week rehabilitation program on lower extremity microvascular reactivity in patients with coronary heart disease (CHD) [56]. Using rSO₂, the effect of a prototype multi-frequency whole-body vibration device, used in the supine position, on local tissue oxygenation in healthy volunteers was assessed. It was shown that therapeutic use of a vibration device induced moderate but significant increases in muscle activation, regional muscle oxygenation and metabolic parameters. These increases were similar to those observed during moderate physical activity, suggesting that whole-body vibration may serve as a suitable approach to combat muscle weakness associated with long-term immobilization [45]. Therefore, in view of the assessment of the possibility of using NIRS in the area of physiotherapy, it seems important that the next step of research in this area should be related to assessment, using the NIRS method, of the nature of changes in rSO₂ and the oxygen consumption in both muscles and tendons in response to physiotherapeutic treatments causing reflex vascular reactions and postulated trophic effect for tissues (e.g., thermotherapy, ultrasound therapy, laser therapy).

In the study group, gender differences were found in some ROIs (Table 3). The SenSmart X-100 NONIN system used in our study uses four wavelengths of near-infrared light. The addition of the third and fourth wavelengths is intended to improve the accuracy of reporting the actual percentage of oxidized hemoglobin in target tissues. The additional wavelengths compensate for the influence of tissue factors that reduce the accuracy of the measurements and allow for the minimization of the influence of interindividual factors (age, weight or skin color) on the result [57,58], which is extremely important in creating referential values. Nevertheless, in the results presented, we observed significantly lower rSO₂ values in the older group compared to the younger group (Table 2) and showed a negative correlation with age (Table 5). This indicates a decrease in rSO₂ values with age, which is confirmed by the source data [58].

Obesity and the related thickness of the adipose tissue above the measurement site also seem to be important factors contributing to the reduction of rSO₂ values (Table 5). In our own research, we showed a negative correlation between rSO₂ and PBF. In areas where fat tissue does not accumulate (carpal tunnel and ankle joint), this correlation was positive but very weak and not statistically significant. Only in the Achilles tendon area did the rSO₂ value show a positive correlation (p < 0.01) with PBF.

5. Limitations of the Study

The authors are aware of the limitations of the presented research. First of all, the research was carried out using one type of device, and comparing data from different devices is a challenge due to the lack of standardization of signal processing and acquisition within the NIRS method. Although the assessment of the fat tissue content in the subjects was performed using the gold standard for assessing body composition (DXA method), it could also be attempted to assess the thickness of the skin folds in the place where the NIRS probe is applied. It is a simple and widely available anthropometric method of assessing nutritional status. This could improve research by assessing the impact of the thickness of subcutaneous fat tissue on the result and by making it possible for subsequent researchers to refer to these data, as well as in clinical assessment, where rSO₂ values are assessed as an indicator of patients’ condition. The last but significant limitation is the relatively small size of the group and the differences in the number of women and men, which did not allow for a clear assessment of the impact of gender.

6. Novelty and Perspective

The novelty of the presented research is based on two aspects that have not been analyzed so far in studies using the NIRS method to assess rSO₂. First, rSO₂ was assessed in 18 independent areas on both sides of the body in 70 healthy people aged 18–30 and elderly people over 60 years of age. This provided the basis for determining rSO₂ reference values in physiological conditions in young and old women and men. The demonstrated significant interarea differences in rSO₂ values under physiological conditions confirm the needs to refer to them in the assessment of pathological changes. Moreover, for the first time the areas where the rSO₂ value decreases with age and increasing fat tissue content have been identified.

Based on the implementation of the goal of our research in the longer term, we would like to determine whether it is possible to consider measuring regional tissue saturation using the NIRS method to assess the reactivity of microcirculation during hyperemia-stimulating physiotherapy in muscles and tendons. Additionally, to clarify the methodology, it seems necessary to assess the influence of gender and the skin fold thickness at the site of application of the NIRS probe on the accuracy of NIRS measurements in patients with different skin pigmentation. It is also necessary to expanding the sample size, of both women and men.

Moreover, NIRS may become an element of computer-aided diagnostics (CAD) in the future. CAD, using data-processing techniques and statistical analyses, is an advanced medical diagnostic tool that is becoming more and more accepted in the medical community. CAD systems have proven to be useful, among others, for early detection of breast cancer [59], acute lymphoblastic leukemia [60] or congestive heart failure [61]. A promising prospect is the use of artificial intelligence (AI) and machine learning in combination with NIRS for modern diagnostics and monitoring of the patient’s health. Although the application of AI to NIRS data has not yet been widely researched, the literature on the subject includes studies on the detection of cerebral ischemia [62], pain imaging [63] or the detection of cognitive load [64,65]

7. Conclusions

The rSO₂ values recorded with the NIRS method at rest are area-specific in young and elderly healthy subjects. This is indicated by the presence of significant differences between areas but no differences between the corresponding ROIs on opposite sides of the body. Age and the content of adipose tissue are factors that may significantly reduce the rSO₂ value. Therefore, referring to normative values for rSO₂ in clinical trials should consider the study area and individual factors such as age and fat tissue content. Having normative data for selected ROIs for healthy subjects seems useful for monitoring patients during surgery, especially those related to the cardiovascular system, where rSO₂ can be used to monitor not only brain perfusion but also other areas sensitive to hypoxia. The changes in oxygen saturation, both within the muscles and tendons, can be non-invasively measured in vivo with NIRS technics, which indicates the usefulness of this method in monitoring tissue repair during the rehabilitation treatment.