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Biology 2023, 12, 60. https://doi.org/10.3390/biology12010060 www.mdpi.com/journal/biology
Article
Effects of Near-Infrared Light on Well-Being and Health in
Human Subjects with Mild Sleep-Related Complaints: A
Double-Blind, Randomized, Placebo-Controlled Study
Marina Cecilia Giménez 1,2,*, Michelle Luxwolda 1,2, Eila G. Van Stipriaan 1,3, Pauline P. Bollen 1, Rieks L. Hoekman 1,
Marthe A. Koopmans 1,2, Praveen R. Arany 4, Michael R. Krames 5,6, Anne C. Berends 5, Roelof A. Hut 2
and Marijke C. M. Gordijn 1,2
1 Chrono@Work B.V., 9743 AD Groningen, The Netherlands
2 Groningen Institute for Evolutionary Life Sciences, University of Groningen,
9747 AG Groningen, The Netherlands
3 Department of Biology, Utrecht University, 3584 CS Utrecht, The Netherlands
4 Department of Oral Biology, Surgery and Biomedical Engineering, University at Buffalo,
Buffalo, NY 14214, USA
5 Seaborough Life Science, 1098 XG Amsterdam, The Netherlands
6 Arkesso LLC, Palo Alto, CA 94306, USA
* Correspondence: marina.gimenez@chronoatwork.com
Simple Summary: In Western societies, people spend most of their waking hours indoors, exposing
themselves to virtually no sunlight. Natural sunlight contains all visible and non-visible spectral
characteristics of light. Both play key roles in human health and well-being. In this particular con-
text, the non-visible near-infrared light has been shown to be beneficial for a wide range of condi-
tions. In the present study, we investigated the effects of morning exposure to near-infrared light
five days per week for four consecutive weeks in a group (n = 56) of healthy individuals with mild
sleep complaints. We observed consistent positive effects on several aspects of well-being and
health but not on sleep or circadian rhythms. The benefits were only visible in the winter months,
when sufficient exposure to sunlight is more challenging. The present study investigated rather
low-energy light levels, which would allow for relatively easy incorporation of such technology into
a household or personal appliances. Because of people’s indoor lifestyle and the need for more
healthy buildings, the current results may open new ways of creating an optimal environment for
a healthier society by preventing some negative effects produced by the lack of sunlight.
Abstract: Modern urban human activities are largely restricted to the indoors, deprived of direct
sunlight containing visible and near-infrared (NIR) wavelengths at high irradiance levels. Thera-
peutic exposure to doses of red and NIR, known as photobiomodulation (PBM), has been effective
for a broad range of conditions. In a double-blind, randomized, placebo-controlled study, we aimed
to assess the effects of a PBM home set-up on various aspects of well-being, health, sleep, and cir-
cadian rhythms in healthy human subjects with mild sleep complaints. The effects of three NIR light
(850 nm) doses (1, 4, or 6.5 J·cm−2) were examined against the placebo. Exposure was presented five
days per week between 9:30 am and 12:30 pm for four consecutive weeks. The study was conducted
in both summer and winter to include seasonal variation. The results showed PBM treatment only
at 6.5 J·cm−2 to have consistent positive benefits on well-being and health, specifically improving
mood, reducing drowsiness, reducing IFN-γ, and resting heart rate. This was only observed in win-
ter. No significant effects on sleep or circadian rhythms were noted. This study provides further
evidence that adequate exposure to NIR, especially during low sunlight conditions, such as in the
winter, can be beneficial for human health and wellness.
Keywords: photobiomodulation; near-infrared; sleep; human clinical trial; immune system; heart
rate; mood; lighting
Citation: Giménez, M.C.; Luxwolda,
M.; Van Stipriaan, E.G.; Bollen, P.P.;
Hoekman, R.L.; Koopmans, M.A.;
Arany, P.R.; Krames, M.R.; Berends,
A.C.; Hut, R.A.; et al. Effects of
Near-Infrared Light on Well-Being
and Health in Human Subjects with
Mild Sleep-Related Complaints: A
Double-Blind, Randomized, Placebo-
Controlled Study. Biology 2023, 12,
60. https://doi.org/10.3390/
biology12010060
Academic Editor: Cristiano Bertolucci
Received: 7 November 2022
Revised: 14 December 2022
Accepted: 22 December 2022
Published: 29 December 2022
Copyright: © 2022 by the author. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distrib-
uted under the terms and conditions of
the Creative Commons Attribution
(CC BY) license (https://creativecom-
mons.org/licenses/by/4.0/).
Biology 2023, 12, 60 2 of 24
1. Introduction
In Western societies, people spend about 85% of their waking hours indoors [1], de-
priving themselves of exposure to direct sunlight. In temperate regions, outdoor sunlight
can reach over 100,000 lux on a cloudless midday. Natural sunlight contains a broad spec-
trum of wavelengths ranging from far infrared (>2000 nm) to ultraviolet (UV 280–400 nm).
On the other hand, indoor light intensities barely reach 500 lux and often contain only
wavelengths within the visible range (420–740 nm). Moreover, modern window glazing
(low-e glass coating) effectively blocks all wavelengths outside the visible range, espe-
cially NIR light, to improve building insulation.
Light has key roles in human health and wellness. Besides its role in enabling human
vision, a direct role of UV-B (280–320 nm) in mediating vitamin D metabolism to maintain
bones and teeth, as well as regulating inflammation and immune functions, has been well
established [2,3]. Exposure to visible light is also known to affect sleep-wake rhythms,
sleep quality, alertness, mood, and performance [4]. It has been reported that near-infra-
red (NIR 750–1100 nm) light accounts for about 54% of the solar radiation reaching the
earth and is assumed to play an essential role in sustaining life on our planet [5], for in-
stance, by improving health [6]. Studies on the exposure to red and NIR light, termed
photobiomodulation (PBM) therapy, can be traced back to the 1960s, when its benefits on
wound healing and the stimulation of hair growth were first observed [7]. Since then, PBM
therapy has been reported to be effective for the treatment of a variety of conditions, such
as wound healing, reducing inflammation or pain, and even treating depression [6,8–10].
In 2002, the U.S. FDA approved PBM treatment for pain relief in cases of head and neck
pain, arthritis, and carpal tunnel syndrome [11]. More recently, PBM therapy has been
recommended by the WALT/MASCC/ISOO guidelines as a standard of care for managing
oral mucositis following oncotherapy such as chemo, radiation, or transplants [12–14].
The effectiveness of PBM is hypothesized to be related to the wavelength, dose, and
pulse characteristics following a biphasic dose-response curve [15]. PBM is a non-thermal
process involving endogenous chromophores eliciting photophysical (i.e., linear and non-
linear) and photochemical events at various physiological levels [8]. The most popular
mechanism for PBM effects involves its actions on mitochondrial metabolism, cell mem-
brane photoreceptors or transporters, and activation of extracellular latent growth factor,
TGF-1 [9,10,15–25]. Most applications of PBM focus on the local effects on tissue that is
directly exposed to far-red or NIR light. However, indirect systemic effects of PBM on
wound-healing, lung inflammation, and Parkinson’s disease have been demonstrated
[18,26–28]. The deeper skin penetration of NIR compared to other visible light (red or
blue) might contribute to the broader beneficial responses [29]. Chronic sleep deficits
and/or circadian misalignment have been hypothesized to contribute to mitochondrial
dysfunction [30,31]. These disruptions have been correlated with an increased risk for car-
diovascular problems and a dysregulated immune system [32–35]. Given the effects of
PBM on stimulating mitochondrial function, its potential therapeutic roles via systemic
effects could benefit people with mitochondrial dysfunction afflicted with a broad range
of diseases.
Decades of developments in the LED industry have finally provided NIR-LEDs with
the adequate power and energy efficiency to provide adequate daylight-like exposure
within standard indoor lighting infrastructure (see patent in the patent section). This
study exploits these developments to explore the systemic effects of exposure to 850 nm
NIR light in people suffering from mild sleep problems (i.e., sleep deficit and/or reduced
sleep quality with clear complaints during daytime) in a double-blind, placebo-controlled
study. Several outcomes within three categories were explored: health, well-being, and
sleep. We hypothesized that PBM would have a positive effect compared to the placebo
treatment and investigated the lowest effective dose in routine lighting for optimal thera-
peutic benefits.
2. Methods
Biology 2023, 12, 60 3 of 24
2.1. Clinical Study Design
A double-blind, placebo-controlled study was conducted at participant’s homes.
Each participant was allocated by stratified randomization to one of four conditions: pla-
cebo, low, medium, or a high dose based on age, chronotype, sleep quality, drowsiness,
depression, sleep duration, and sleep deficit. NIR illumination of the skin of the face, neck,
and hands was performed on subjects seated behind a desk. The study took place over
three periods of the year, evenly distributed over summer and winter: January 2021–
March 2021 (winter group), halfway April–July 2021 (summer group), and halfway No-
vember–December 2021 (winter group). Participants were recruited via advertisements
and flyers. Inclusion criteria were: an average sleep duration per week ≤ 6.5 h, [36], and/or
showing an accumulated sleep deficit of at least 1 h during the week [37], and/or reduced
sleep quality (Pittsburgh Sleep Quality Index, PSQI > 5, [38]) with clear drowsiness com-
plaints during daytime; Epworth Sleepiness Scale, ESS > 5 [39] or a mild depression score
≥ 13 and < 19; Beck’s Depression Inventory, BDI [40]. Exclusion criteria were: BDI > 19,
pregnancy, menopause complaints, use of immunosuppressants, shift work, travel over
more than 2 time zones, high alcohol intake (more than 4 units for men and more than 3
units for women per day, for more than 5 days in the past month), use of medications that
are known to interfere with sleep, alertness, the biological clock and/or light sensitivity,
and high levels of caffeine intake (5 or more cups per day). The study took place in the
participant’s home and/or workplace for 4 consecutive weeks, mainly during home-work
regulations by the government to prevent the spread of COVID-19. All study procedures
were approved by the Medical Ethical Research Committee of the University Medical
Centre Groningen (NL74857.042.20), The Netherlands. The procedures are in accordance
with the Declaration of Helsinki (2013) and registered at the Netherlands Trial Register
(#NL8800). All participants gave written informed consent and received financial com-
pensation for their participation.
2.2. PBM Treatment
2.2.1. Device
The PBM module was incorporated into a regular desk lamp (IKEA Ypperlig) (Figure
1A). It consisted of a wooden box with high-power 850 nm Lumileds NIR-LEDs (L1I0-
0850090000000, Schiphol, The Netherlands) with a beam angle of 90 degrees. It was ori-
ented in order to assure a 1sr beam that covered the user’s face, neck, and hands on the
desk, with tissue surface irradiance of 5 mW·cm−2. This resulted in an estimated total illu-
minated area of approximately 1850 cm2 and of 1600 cm2 for males and females, respec-
tively, as calculated using https://msis.jsc.nasa.gov/ (accessed on 7 December 2022), vol-
ume 1, section 3. Subjects were instructed not to cover the skin of their face, neck, and
hands and not to use any skin products prior to treatment.
The module was further equipped with a distance and presence sensor (VL53L1X-
SATEL, STMicroelectronics, Geneva, Switzerland), that allowed for NIR intensity adjust-
ment to maintain a peak irradiance of 5 mW/cm2 on the skin as the user leaned forward
or backward. Furthermore, if subjects came too close to the device (<20 cm), the radiation
was switched off for safety reasons and a green LED would signal that an error had oc-
curred, in which case participants knew they had to adjust their position. At the back of
the module there was an ambient vertical lux sensor (BH1750FVI, ROHM Co., Ltd., Kyoto,
Japan).
Biology 2023, 12, 60 4 of 24
2.2.2. Dose
The LEDs for PBM in this study were operated in pulsed mode with their correspond-
ing duty cycle [15,41,42]. The PBM dose was established by changing frequency/duty cy-
cle and the duration of the PBM resulting in doses of 0 J·cm−2, 1 J·cm−2, 4 J·cm−2, and 6.5
J·cm−2 (Table 1, Figure 1B). Other SI units often used to describe PBM dose are shown in
Table 1. Dose and timing were programmed into the device, so that no user intervention
was necessary. The NIR stimulation was not visible to the eye nor was it felt on the skin
as heat. Strips of red (633 nm) LEDs (OSRAM, LS R976-NR-1, Munich, Germany) were
blinking in low intensity at 10 Hz frequency to further prevent the possibility of visually
noticing the NIR stimulation and to provide some user feedback regarding the device be-
ing “on’’.
Participants received a PBM module and were asked to sit in front of it from 9:30 am
until 12:30 pm 5 times per week for four consecutive weeks. The PBM module switched
on (9:30 am) and off (12:30 pm) automatically.
Figure 1. (A) PBM treatment module used in this study. The PBM module is incorporated in a desk
lamp and its power is controlled by the driver at the table. The strips of low-power red LEDs at the
front of the module were turned on in all conditions. The distance sensor (large circle) is shown. The
smaller circle in the upper left corner is a green LED that provides feedback on the distance of the
user. (B) A schematic overview of the different PBM dose durations all timed relative to the offset
of the PBM session.
Table 1. Dosimetry overview. # refers to ‘number of photons’.
Dose Duration Duty Factor Peak Irradiance
Photonic Dose
Molar Dose
J·cm–2 m s % mW. cm–2 # .cm–2 µmol. cm−2
Low 1.0 42 2520 8% 5 4.3 × 1018 7.2
Med 4.0 168 10,080 8% 5 1.7 × 1019 28.6
High 6.5 180 10,800 12% 5 2.8 × 1019 46.0
2.3. Outcomes Assessment
Actigraphy measurements (Motionwatch 8, Camntech, Fenstanton, UK, and Fitbit
Versa 3, Fitbit Inc., San Francisco, California, USA) started one day before the first PBM
session. During the evenings of the baseline measurements scheduled 3 days prior to the
first PBM session, as well as after the first and second session (day 1 and 2) and after 10
and 20 PBM sessions (week 2 and 4), the participants performed the following actions
Biology 2023, 12, 60 5 of 24
(Figure 2): filled out questionnaires regarding mood (1−10 Likert scale) and drowsiness
(0−24 Epworth Sleepiness Scale (ESS, [39]); collected hourly saliva samples from 5 h before
habitual sleep onset until 1 h after for assessment of Dim Light Melatonin Onset (DLMO)
and cortisol; collected urine between 8 pm (after emptying the bladder) and 8 am for as-
sessment of total night-time melatonin production. Furthermore, during the first 2 PBM
sessions skin temperature was measured using iButtons.
Only at baseline and after 2 and 4 weeks, the following additional assessments were
performed in the evening: questionnaires regarding sleepiness (1−9 scale, Karolinska
Sleepiness Scale, KSS, [43]), subjective sleep quality (0−10 Likert scale), sleep complaints
(0−21 scale, Pisburgh Sleep Quality index, PSQI, [38]), WHO subjective performance
(0−10 scale), and need for recovery (0−100 scale, [44] ) were filled out. At daytime, blood
was collected for the assessment of interferon gamma (IFN-γ) and tumor necrosis factor
alpha (TNF-α).
Three assessments were only performed at baseline and after 4 weeks: blood collec-
tion for the assessment of vitamin D as an indication of exposure to outdoor light, hair
sample collection for assessment of accumulated cortisol, and completing the BDI ques-
tionnaire.
Figure 2. Schematic overview of the study design and performed measurements.
2.3.1. Saliva Collection
Participants were carefully instructed about the requirements for collecting saliva.
No chocolate, bananas, artificially colored sweets, coffee, or black tea were allowed as well
as brushing teeth with toothpaste. Eating and drinking were not allowed in the 30 min
before the collection of saliva, and max. 10 min before each sample subjects were in-
structed to rinse their mouths with water. Subjects were also asked to expose themselves
to as little light as possible by keeping the curtains closed, using only small light bulbs,
dim light, decreasing brightness, using blue-blocking filters on screens, and wearing sun-
glasses inside, commencing 1 h before the first sample. Watching TV was allowed at a
distance of ≥ 2 m. Postural changes were not allowed during the 5 min period before and
during saliva collection. Saliva was collected by the participants using Salivette® (Sar-
stedt™ Ltd., Nümbrecht, Germany) and stored overnight at approximately 4 °C. Samples
were collected within 2 days, the Salivettes were centrifuged, and the saliva was trans-
ferred to 2 mL Eppendorf tubes and stored at −80 °C.
Last day W2
Day 1 W1
Day 2 W1
Last day W4
Baseline
W1 W2 W3 W4
Morning Evening/night
Legend:
W1-4 = Week 1-4
Motionwatch &
Fitbit
Blood
withdrawn
Saliva and urine
collection
Questionnaires
PBM module
Hair sample
ibuttons
Biology 2023, 12, 60 6 of 24
2.3.2. Dim Light Melatonin Onset Assessment
On completion of the study, a double-antibody radioimmunoassay (RIA) was per-
formed to assess melatonin concentration levels (Direct Saliva Melatonin kit, NovoLytiX
GmbH, Switzerland; intra-assay variation: 20.1% and 4.8%; inter-assay variation: 16.7%
and 8.4% for low and high concentration samples, respectively). Dim light Melatonin On-
set (DLMO) was assessed for the first time when melatonin concentrations exceeded the
3 pg/mL threshold upon linear interpolation of subsequent melatonin values.
2.3.3. aMTs6 Assessment
Urine was collected to measure degradation of melatonin by the production of 6-
sulfatoxymelatonin (aMTs6) throughout the night between 8 pm and 8 am. It was stored
at approximately 4 °C. Within 2 days, the urine was received by the research institute. The
total volume was measured, and samples were transferred to 2 mL Eppendorf tubes and
stored at −80 °C. On completion of the study, an enzyme-linked immunosorbent assay
(ELISA) was performed (6-Sulfatoxymelatonin kit, NovoLytiX GmbH, Switzerland; intra-
assay variation: 9.7% and 5.3%; inter-assay variation: 15.3% and 8.4% for low and high
concentration samples, respectively).
2.3.4. Cortisol Assessment
Cortisol analysis was carried out using the same saliva samples as used for melatonin
analysis. The samples collected 3 and 4 h before bedtime were pooled (3.5 h before bed-
time), as well as the samples collected 1 h before and at bedtime (0.5 h before bedtime).
On completion of the study, a double-antibody radioimmunoassay (RIA) was used to as-
sess cortisol concentrations levels (CORT-CT2 radioimmunoassay kit, Cisbio Bioassays,
France; intra-assay variation: 3.9% and 3.2%; inter-assay variation: 7.6% and 5.1% for low
and high concentration samples, respectively). Hair cortisol was collected by cutting a
pencil-sized hair strand from the scalp at the back of the head and was stored in tinfoil at
room temperature. On completion of the study, an online-solid phase extraction (SPE)
combined with a fully validated isotope dilution liquid chromatography tandem mass
spectrometry (LC–MS/MS) was performed (lab-developed, Department of Laboratory
Medicine, Special Chemistry at the University Medical Center Groningen, The Nether-
lands [45]; intra-assay variation: 9.3% and 4.3%; inter-assay variation: 6.1% and 6.0% for
low and high concentration samples, respectively). The lower limit of quantitation for hair
cortisol was 0.70 pg/mg hair.
2.3.5. Cytokines and Vitamin D Assessments
Two blood samples of 4 mL each were collected in EDTA-coated tubes. Samples were
centrifuged, and plasma supernatant was recovered and stored in Eppendorf tubes at −80
°C. On completion of the study, an enzyme-linked immunosorbent assay (ELISA) was
performed to assess IFN-γ and TNF-α concentration levels (Quantikine Human IFN-γ
Immunoassay kit, and Human TNF-α Immunoassay kit, Bio-Techne Ltd., Abingdon, UK;
intra-assay variation: 4.6% and 2.0% for IFN-γ and 2.2% and 1.9% for TNF-α; inter-assay
variation: 9.8% and 7.2% for IFN-γ and 6.7% and 6.2% for TNF-α for low and high con-
centration samples, respectively).
Plasma 25-hydroxyvitamin D3 levels were determined using isotope dilution–
online-solid-phase extraction liquid chromatography–tandem mass spectrometry (ID-
XLC–MS/MS) (lab-developed, Department of Laboratory Medicine, Special Chemistry at
the University Medical Center Groningen, The Netherlands; inter-assay variation: 4.5%
and 6.8% for low and high concentration samples, respectively).
2.3.6. Skin Temperature
Skin temperature was measured using iButtons (DS1922L, resolution: 0.0625 °C,
Maxim Integrated, San Jose, CA, USA) on the first two days of PBM exposure. Participants
Biology 2023, 12, 60 7 of 24
were instructed to apply 7 iButtons: one on the forehead, two on the dorsal side of both
middle fingers, 2 on the distal side of both claviculae, and two on the inner side of both
ankles, and secured with Fixomull Stretch tape (BSN Medical B.V., Almere, The Nether-
lands). The iButtons were worn during the 3.5 h of PBM exposure + time needed for ques-
tionnaires (09:15 u–12:45 u). The same iButton was used on the same skin location for two
consecutive days. Data were collected after the second PBM session using OneWire
Viewer software.
2.3.7. Composite Scores for Well-Being, Health, and Sleep Quality
Composite scores were calculated only from those outputs that were obtained after
2 and 4 weeks. Outputs were classified into three categories, and a composite score was
calculated. The categories were: 1) well-being, including all questionnaire-related outputs,
2) health, including immune outputs, cortisol at bedtime, and resting heart rate (Fitbit),
and 3) sleep quality, including actigraphy-derived sleep fragmentation, PSQI, the general
sleep score, and minutes of deep sleep (Fitbit). Although the accuracy of the Fitbit-derived
sleep parameters compared to polysomnography is still under debate, the within-subject
changes are of interest for the current study [46].
To calculate the composite scores, each variable was Z-transformed, including all in-
dividual values at baseline, week 2, and week 4. For each subject at each timepoint, the 3
composite scores were calculated as follows: (1) the well-being composite score was the
sum of each individual’s Z-transformed value of mood, drowsiness, sleepiness, need for
recovery, and subjective performance, with each value being positive for better mood, less
drowsy, less sleepy, less need for recovery and better performance; (2) the health compo-
site score was the sum of each individual’s Z-transformed value of TNF-ɑ, IFN-γ, cortisol
at bedtime (selected in view of a possible relationship with stress at bedtime), and resting
heart rate value, again with a positive score meaning good health: less TNF-ɑ, less IFN-γ,
lower cortisol at bedtime and lower resting heart rate; (3) the sleep quality composite score
was the sum of each Z-transformed value of PSQI, sleep fragmentation, minutes of deep
sleep and sleep score, with a higher sleep quality score meaning good sleep: high sleep
quality, low PSQI, low sleep fragmentation score on the actigraphy, high amount of deep
sleep minutes and high general sleep score on the Fitbit.
2.4. Data analysis
All statistics were performed in R (R Core Team, 2021; version: 4.1.0), using the most
recent shell of R studio (version: 2022.02.0). ANOVA or T-tests were used to assess differ-
ences between groups’ demographics, light exposure during PBM sessions, and skin tem-
perature data. Light intensity values from the light sensor at the back of the PBM module
were transformed to a log(10) scale and averaged for each participant throughout the PBM
session. Skin temperature data were z-transformed to the mean and SD of all conditions
and all individuals and averaged throughout the first 40 min after the start of the PBM
treatment (i.e., maximum PBM duration in the 1 PBM condition) for each individual. In
the Supplementary Materials, figures for the main items of well-being, health, and sleep
are shown with the raw data (Figures S1–S3). Given the in-between subjects design, re-
sponses were normalized to each individual’s baseline. This was done by subtracting
baseline values of each variable from their corresponding output after a given time; after
1 or 2 days (short time-frame), as well as after 2 and 4 weeks (long time-frame). The anal-
ysis followed the subsequent structure: (1) long time-frame data were assessed by means
of a composite score, and (2) if a significant effect was observed, the single items contrib-
uting to the composite score were explored. For the composite score as well as the single
items, the interaction effect between PBM dose and time (week 2 vs. week 4) was first
assessed by means of a mixed ANOVA, with dose and season as between factors and time
as within factor. If no significant interaction with time was observed, an accumulated av-
erage of the difference between week 2 and baseline and of week 4 and baseline was used
for further analysis. A similar approach was taken for the analysis of the short time-frame
Biology 2023, 12, 60 8 of 24
data (day 1 vs. day 2). No composite scores were calculated for the short time-frame data.
Outputs are expressed as main effects of the factor PBM dose compared to placebo, as
well as the factor PBM dose for the winter and summer groups, and the interaction effect
between PBM dose and season. Motionwatch as well as Fitbit data were collected on a
daily basis. For these outputs, an overall effect throughout all days as well as a delta for
the first and second day relative to baseline were analyzed. Based on our a priori hypoth-
esis and that the treatment (PBM dose) has more than 2 levels, the effects of dose and the
potential modifying effects of season were tested as treatment contrasts by means of linear
models. Linear models allow for the use of all data available per group as well as for con-
struction of mixed-effects models. For the linear models, factors included were dose with
4 levels, season with 2 levels, and the interaction between dose and season. As secondary
analysis, the effects of adding BMI and age as factors to the model and their interaction
with dose were studied. Only significant interactions with BMI and age are reported. For
the daily outputs of Motionwatch and Fitbit, the model also included days as a factor and
its interaction with dose. Only the days in which the PBM module was used were in-
cluded. Critical two-sided significance level alpha was 0.05 for all statistical tests. Due to
the limited number of subjects per group (8 or less per season, Table 2), trends with an
alpha up to 0.1 are also reported.
Table 2. Demographics of the individuals in the different groups for summer and winter together.
Except for the number of male and female participants, all values are shown as average (standard
error of the mean, SEM is noted between brackets). Abbreviations used: PSQI = Pittsburgh Sleep
Quality Index, ESS = Epworth Sleepiness Scale, BDI = Beck’s Depression Inventory, BMI = Body
mass index, M= Male, F = Female.
0 J·cm−2 1 J·cm−2 4 J·cm−2 6.5 J·cm−2 Significance
Number (M:F) 13 (4:9) 14 (5:9) 15 (7:8) 14 (6:8)
Age (y) 37.9 (3.4) 38.2 (3.3) 38.7 (5.8) 37.4 (3.5) ns
Chronotype (h) 4.1 (0.4) 4.2 (0.4) 4.3 (0.4) 4.3 (0.5) ns
Sleep duration (h) 7.0 (0.4) 7.3 (0.3) 7.4 (0.3) 6.9 (0.3) ns
Sleep deficit (h) 1.4 (0.4) 1.6 (0.3) 1.0 (0.2) 1.5 (0.5) ns
PSQI 10.7 (1.0) 10.4 (1.1) 9.8 (0.9) 10.6 (0.7) ns
ESS 8.6 (1.1) 7.7 (1.5) 8.5 (1.3) 9.6 (1.2) ns
BDI 10.5 (1.2) 11.1 (1.6) 12.3 (1.4) 11.1 (1.5) ns
BMI (kg) 25.4 (1.1) 25.3 (1.5) 24.7 (1.1) 24.6 (0.9) ns
3. Results
Out of originally 62 subjects who were selected to participate, 56 completed the study
(22 males and 34 females, age: 25−64, Caucasian: 51, non-Caucasian, non-Asian: 4, Asian:
2). Five participants cancelled their participation in an early stage, one due to illness not
related to the study, one because of a pregnancy, and three because of personal circum-
stances. One participant was excluded from the analysis due to non-compliance. No sig-
nificant differences between groups were observed, indicating a good a priori matching
(Table 2).
To examine any variances in the groups between seasons, the winter and summer
groups were assessed separately as well (Table S1). No significant differences in any of
the parameters were observed between the groups per season. A significant difference in
chronotype and sleep deficit in the groups between seasons were noted. The winter season
group showed an earlier chronotype (3.9 h ± 0.2 SEM) and less sleep deficit (0.9 h ± 0.1
SEM) than the summer season group (4.6 h ± 0.2 and 1.9 h ± 0.3, for chronotype and sleep
deficit, respectively).
Analysis of the ambient light levels indicated that environmental indoor light levels
(average 1.82 ± 0.56 SD log lux) did not differ significantly among the groups (F3,48 = 0.29,
p = 0.83) despite the evident seasonal variation (1.96 ± 0.56 SD log lux summer and 1.69 ±
0.54 SD log lux winter). There was no significant interaction effect of the light intensity
Biology 2023, 12, 60 9 of 24
between the PBM dose and season (F3,48 = 0.2, p = 0.89), suggesting that PBM treatment
effects cannot be explained by differences in environmental light exposure during the
PBM sessions (Figure S4A). These findings were further expanded to include outdoor
light exposure, as shown by the changes in vitamin D throughout the study. While there
was a significant effect of the season with a larger increase of vitamin D levels during the
4 week period in the summer (7.8 ± 11.2 SD) compared to a decrease (−4.2 ± 8.5 SD) in the
winter (F1,48 = 18.96, p < 0.001), there were no significant differences among groups (F3,48 =
0.89, p = 0.45), nor was there a significant interaction between season and NIR dose (F3,48 =
0.56, p = 0.64; Figure S4B).
3.1. Effects of PBM Treatment on Well-Being
For the composite score of well-being, no significant interaction between The PBM
dose and time was observed (F3,48 = 0.46, p = 0.71). The cumulative average change in well-
being over 2 and 4 weeks showed no significant effect of PBM treatment (−0.15 ± 1.15,
−0.87 ± 1.13, 0.94 ± 1.15, for the 1, 4, and 6.5 PBM doses, respectively) compared to the
placebo (1.98 ± 0.83, all p > 0.4). However, the analysis of the season factor revealed for the
winter group a significant improvement in the 6.5 PBM group (3.65 ± 1.41, p < 0.01) com-
pared to the placebo (0.46 ± 1.03).
Conversely, the summer group did not demonstrate any significant effect of the PBM
treatments (−0.44 ± 1.52, −1.60 ± 1.51, −2.41 ± 1.57, for the 1, 4 and 6.5 PBM doses, respec-
tively) compared to the placebo (3.77 ± 1.1 all p > 0.2). This is further supported by the
significant interaction effect between the 6.5 PBM dose and the seasons (p < 0.001), indi-
cating that the beneficial effects of PBM treatment on the participant’s well-being in the
6.5 J·cm–2 treatment condition are only present in the winter (Figure 3A, Table S2A). The
main effect of the seasons was also significant, showing an overall increase of 3.30 points
(± 1.52 SEM, p < 0.001) over the four weeks on the well-being score in the summer group,
independent of PBM condition.
Figure 3. Change in composite score for winter and summer separately (average of the difference
between week 2 and baseline and of week 4 and baseline) for (A) well-being, (B) health, and (C)
sleep. Significance codes: ** p < 0.01, * p < 0.05, ns: not significant. Sample sizes per condition are
shown.
The individual items contributing to the composite well-being are elaborated below.
Mood: no significant interaction between the PBM dose and time was observed (F3,48
= 0.35, p = 0.89). The cumulative average over 2 and 4 weeks showed a tendency for an
improvement of mood over time in the 6.5 PBM group of about half a point (±0.30 SEM, p
< 0.1), compared to a smaller improvement in the placebo group (0.17 ± 0.22). The analysis
of the season revealed in the winter group a significant improvement of mood over time
in the 6.5 PBM group (1.46 ± 0.34, p < 0.001) compared to a small decrease in mood in the
Biology 2023, 12, 60 10 of 24
placebo group (−0.48 ± 0.25), which was also reflected in the significant interaction be-
tween the 6.5 PBM dose and season (p < 0.001). In summer, on the other hand, a small
deterioration of mood over time was observed for the 4 PBM condition (−0.79 ± 0.37, p <
0.05) compared to a small improvement in the placebo group (0.94 ± 0.27) (Figure 4A,
Table S2B). Overall, independent from condition, mood improved in the summer (1.4 ±
0.37 SEM) more than in the winter, as shown by the significant effect of the season factor
(p < 0.001).
On a shorter time-frame (i.e., day 1 and day 2), a significant interaction effect between
the factors PBM dose and time was found (F1,48 = 3.9, p < 0.05). Namely, a tendency for an
improvement of mood over time was observed in the 6.5 PBM group (0.52 ± 0.31, p < 0.1),
compared to the placebo group (−0.19 ± 0.22) after 1 day, while no effects of the PBM dose
(−0.33 ± 0.31, −0.14 ± 0.31, 0.33 ± 0.31, for the 1, 4, and 6.5 PBM dose, respectively) compared
to the placebo (0.29 ± 0.22, all p < 0.3) was observed on the second day. The analysis of the
season factor revealed that these effects were only observed in the winter group, in which
mood improved significantly over time in the 6.5 PBM group (1.01 ± 0.39, p < 0.05) after
one PBM stimulation compared to the placebo group (−0.37 ± 0.29). A trend for a signifi-
cant interaction between the 6.5 PBM dose and the season was observed (p < 0.1). No sig-
nificant effects of the PBM treatment were observed after two stimulations. In the summer
group, no effects of PBM treatment were observed for day 1 (0.61 ± 0.42, 0.53 ± 0.43, −0.09
± 0.44, for 1, 4, and the 6.5 PBM dose, respectively) compared to the placebo group (−0.00
± 0.31, all p > 0.2), or day 2 (−0.19 ± 0.45, −0.06 ± 0.45, −0.07 ± 0.47, for 1, 4, and 6.5 PBM
dose, respectively), compared to the placebo group (0.48 ± 0.33, all p > 0.7).
Drowsiness: Examination of drowsiness ratings with the Epworth Sleepiness Scale
revealed a significant interaction effect between the PBM dose, time (long-term), and sea-
son (F3,48 = 3.08, p < 0.05). Irrespective of the season, no effects of the PBM dose were ob-
served after 2 weeks (−0.22 ± 0.95, 0.79 ± 0.94, −1.22 ± 0.96 for the 1, 4, and 6.5 PBM condi-
tion) compared to the placebo group (−0.92 ± 0.69, all p >0.2), or after 4 weeks (−0.03 ± 1.19,
0.48 ± 1.17, −0.95 ± 1.19 for the 1, 4, and 6.5 PBM, respectively) compared to the placebo
group (−1.61 ± 0.86, all p > 0.4). The analysis of the season factor revealed that it is only in
the winter group that drowsiness is significantly reduced in the 6.5 PBM group (−2.57 ±
1.19, p < 0.05) over the first 2 weeks compared to the placebo group (−0.43 ± 0.87). A similar
trend was noted after 4 weeks in the 6.5 PBM group (−2.98 ± 1.55, p < 0.1) compared to the
placebo group (−0.14 ± 1.13). The cumulative average difference over 2 and 4 weeks
showed a significantly larger reduction in drowsiness in the 6.5 PBM group in winter
(−2.78 ± 1.25, p < 0.05) compared to the placebo control group (-0.28 ± 0.91). No effects of
the PBM dose on drowsiness were observed in the summer group (−0.23 ± 1.34, 1.06 ± 1.34,
1.00 ± 1.39) compared to the placebo group (−2.41 ± 0.98, all p > 0.4) (Figure 4B, Table S2B).
Sleepiness: Examination of sleepiness ratings with the Karolinska Sleepiness Scale
showed no significant interaction between the PBM dose and time (F3,48 = 0.69, p = 0.89).
The cumulative average of sleepiness changes over 2 and 4 weeks showed no significant
effect of the PBM dose compared to the placebo (all p > 0.3). The analysis of the season
factor revealed no other significant effects of PBM treatment (Figure 4C, Table S3).
On a shorter time-frame (i.e., day 1 and day 2) no significant interaction between the
PBM dose and time was observed (F3,47 = 1.02, p = 0.35). The cumulative average change in
sleepiness score over 1 and 2 days showed no significant effect of the PBM dose compared
to the placebo (all p > 0.4). The analysis of seasons showed no other significant effects of
PBM treatment (Table S4). The KSS score was missing for one participant during the first
evening in the placebo group.
Need for recovery: No significant interaction effect between the PBM dose and time
was observed for the change in the need for recovery (F3,48 = 0.98, p = 0.41). The cumulative
average over 2 and 4 weeks, irrespective of season, showed no significant effect of the
PBM dose on the need for recovery compared to the placebo (all p > 0.7). The analysis of
seasons showed no other significant effects of the PBM treatment on the need for recovery
(Figure 4D, Table S3).
Biology 2023, 12, 60 11 of 24
The addition of BMI to the model revealed a significant effect in the cumulative av-
erage over 2 and 4 weeks of the 6.5 PBM dose; a larger increase in the need for recovery
over time was observed (145.82 ± 68.81, p < 0.05) compared to the placebo (−105.4 ± 45.0),
as well as an interaction effect between the 6.5 PBM dose and BMI (−5.97 ± 2.71, p < 0.05).
This suggests that the higher the BMI, the less negative impact the 6.5 PBM dose has on
the need for recovery. The analysis of the season factor showed a similar pattern. In the
winter group, there is a tendency for a significant detrimental change in the need for re-
covery over time in the 6.5 PBM condition (141.84 ± 74.83, p < 0.1) compared to the placebo
(−103.70 ± 52.97), as well as an interaction between 6.5 PBM and BMI (−6.09 ± 2.87), by
which the effect is slightly compensated with a higher BMI. Furthermore, in the summer
group, a similar observation was found; a significant increase in the need for recovery in
the 6.5 PBM group (160.82 ± 71.81, p < 0.05) compared to the placebo group (−104.63 ±
47.25) as well as an interaction between 6.5 PBM and BMI (−6.09 ± 2.87) (Table S5).
Subjective performance: No significant interaction effect between the PBM dose and
time was observed in the subjective ratings of performance (F3,48 = 1.53, p = 0.22). The cu-
mulative average over 2 and 4 weeks showed no significant effect of PBM treatment (0.09
± 0.51, −0.11 ± 0.50, −0.36 ± 0.51, for 1, 4, and 6.5 PBM doses, respectively) compared to the
placebo (0.61 ± 0.37, all p > 0.5). The analysis of the season factor showed in the summer,
a tendency of a change in the subjective performance in the 6.5 PBM condition (−1.25 ±
0.73), compared to the placebo (0.83 ± 0.52, p < 0.1) (Figure 4E, Table S2B), suggesting a
worsening of subjective performance. No effects of PBM treatment on subjective perfor-
mance were observed in the winter group for any of the doses (0.21 ± 0.68, −0.68 ± 0.66,
0.32 ± 0.66 for the 1, 4, and 6.5 doses) compared to the placebo group (0.43 ± 0.48, all p >
0.3, Table S2B).
On a shorter time-frame (i.e., day 1 and day 2) no interaction effect between the PBM
dose and time was observed in subjective performance ratings (F3,47 = 1.59, p = 0.34). The
subjective performance score was missing for one participant during the first evening in
the placebo group. Furthermore, the cumulative average over 1 and 2 days, irrespective
of season, showed no significant effect of the PBM treatment on subjective performance
ratings for any of the doses compared to the placebo (all p > 0.42). The analysis of the factor
season revealed no other significant effects of the PBM treatment (Table S4).
Biology 2023, 12, 60 12 of 24
Figure 4. Overview of all individual elements of the well-being composite score, for the summer
and winter separately. (A) mood, (B) drowsiness (ESS), (C) sleepiness (KSS), (D) need for recovery,
and (E) subjective performance. Significance codes: *** p < 0.001, * p < 0.05, # p < 0.1 ns: not significant.
Sample sizes per condition are shown.
Depression: This outcome was not included in the composite score as it was only
assessed at baseline and after 4 weeks (i.e., there was no 2 weeks measurement). No sig-
nificant effect on the change of depression scores was observed for any PBM dose (0.19 ±
1.93, −0.17 ± 1.90, −0.59 ± 1.93) compared to the placebo (−3.69 ± 1.39, all p > 0.8). The anal-
ysis of the season factor showed a trend for an interaction effect between 6.5 PBM and
season (−6.8 ± 3.86, p < 0.1), suggesting a larger reduction in depression scores in response
to PBM treatment in the winter group (Figure S5).
3.2. Effects of PBM Treatment on Health
In the analysis of the composite score ‘health’, no interaction was found between the
PBM dose and time (F3,48 = 0.85, p = 0.47). The cumulative average over 2 and 4 weeks
showed a significantly larger improvement in health over time in the 6.5 PBM group (2.83
± 0.95, p < 0.01) compared to the placebo group (−0.89 ± 0.68). The analysis of the season
factor revealed that in the winter group, the 6.5 PBM dose asserts a significantly larger
improvement over time (3.67 ± 1.26, p < 0.01) compared to the placebo group (−0.66 ± 0.9),
but not in the summer group (1.68 ± 1.40) compared to the placebo group (−1.17 ± 0.99, p
= 0.24) (Figure 3B, Table S2A). The changes in individual items are further elaborated be-
low.
IFN-γ: No interaction was found between the PBM dose and time (F3,48 = 0.72, p =
0.55). The cumulative average over 2 and 4 weeks showed a significantly larger reduction
in IFN-γ concentrations of about 1.7 pg/mL (± 0.71 SEM, p < 0.05) compared to the placebo
(0.27 ± 0.51 pg/mL). The analysis of the season factor revealed only in the winter group a
significantly larger reduction in the 6.5 PBM group of about 3 pg/mL (−2.84 ± 0.92 pg/mL,
(p < 0.01), compared to a small increase in IFN-γ concentrations in the placebo group (0.49
± 0.68 pg/mL) (Figure 5A, Table S2C). No effect of treatment was found in the 6.5 PBM
condition in the summer group (−0.18 ± 1.03 pg/mL) compared to the placebo group (0.01
± 0.73 pg/mL, p = 0.86).
TNF-α: No interaction was found between the PBM dose and time (F3,48 = 0.53, p =
0.66). The cumulative average over 2 and 4 weeks showed no effect of the PBM treatment
on the TNF- α concentration compared to the placebo (all doses p > 0.2). The analysis of
the season factor revealed no further effects of the PBM treatment in either the winter or
summer group (Figure 5B, see Table S3).
The addition of BMI to the model led to the observation of a significant reduction of
about 1 pg/mL of TNF- α in the 6.5 PBM group over time (-1.09 ± 0.47 pg/mL, p < 0.05)
compared to a small increase in the placebo group (0.40 ± 0.30 pg/mL), as well as to a
significant interaction effect between BMI and the dose 6.5 PBM (0.04 ± 0.02 pg/mL, p <
0.05) (Table S5). This means that higher BMI levels prevent to a small extent the decrease
in TNF-α in response to the 6.5 PBM treatment. The analysis of the season factor revealed
that the effect of the 6.5 PBM treatment was present in the winter group (-1.09 ± 0.49
pg/mL), but significantly different compared to the placebo group (0.41 ± 0.35 pg/mL, p <
0.05) as well as in the summer group (−1.16 ± 0.47 pg/mL, p < 0.05) compared to the placebo
(0.41 ± 0.31 pg/mL). Again, an interaction between 6.5 PBM and BMI was observed (0.04
± 0.02 pg/mL, p < 0.05), indicating that higher BMI levels reduce the reduction in TNF-α
levels in response to PBM treatment.
Cortisol 0.5h before bedtime: No interaction effect between the PBM dose and time
was found (F3,46 = 0.68, p = 0.57). The cumulative average over 2 and 4 weeks showed a
significant reduction in cortisol levels at bedtime in the 6.5 PBM condition (−8.05 ± 3.86
nmol/L, p < 0.05) compared to the placebo condition (3.60 ± 2.78 nmol/L). The analysis of
the season factor revealed no further effects of the PBM treatment with any dose neither
Biology 2023, 12, 60 13 of 24
in the winter group (−2.79 ± 5.06, −1.92 ± 5.38, −8.25 ± 5.21 for the 1, 4, and 6.5 PBM doses,
respectively) compared to the placebo group (0.79 ± 3.81 nmol/L, all p > 0.1), nor in the
summer group (−7.46 ± 5.60, −3.90 ± 5.60, −7.31 ± 5.81 for the 1, 4, and 6.5 PBM doses,
respectively) compared to the placebo group (6.87 ± 4.11 nmol/L, all p > 0.2) (Figure 5C,
Table S2C). Two participants (1x 1PBM and 1x 4PBM condition) did not generate enough
material for a cortisol analysis.
The analysis of the effect of the PBM dose on cortisol levels at bedtime on a shorter
time-frame (i.e., day 1 and day 2) did not reveal any significant interaction with assess-
ment time (F3,48 = 1.27, p = 0.29). The cumulative average over 1 and 2 days showed no
significant effects for PBM compared to the placebo (all p > 0.2). The analysis of the season
factor revealed no further effects of the PBM treatment (Table S4).
Cortisol 3.5 h before bedtime: This was not included in the composite score as the
largest expectations were focused on cortisol levels just prior to bedtime and the data
points are closely related. Three participants (1 in 1PBM condition and 2 in 4 PBM condi-
tion) did not generate enough material for cortisol analysis. No interaction effect was ob-
served between the PBM dose and time in analyzing the effect on evening cortisol levels
(F3,45 = 0.64, p = 0.59). The cumulative average over 2 and 4 weeks showed no significant
effects of PBM compared to placebo (all p > 0.3) on the change in the level of evening
cortisol. The analysis of the season factor revealed no further effects of the PBM treatment
in the winter summer groups. See Table S3.
On a shorter time-frame (i.e., day 1 and day 2), the PBM dose and time showed no
significant interaction effect (F3,48 = 0.32, p = 0.81) on the change in evening cortisol levels.
The cumulative average over 1 and 2 days showed a tendency for a reduction in evening
cortisol concentrations over time in the 6.5 PBM condition (−3.03 ± 1.81 nmol/L, p < 0.1)
compared to the placebo (0.65 nmol/L ± 1.30). The analysis of the season factor revealed
no effect of the PBM dose in the winter group (−0.29 ± 2.61, 0.20 ± 2.52, −2.74 ± 2.52 for the
1, 4, and 6.5 PMB doses, respectively) compared to the placebo group (0.43 ± 1.84 nmol/L,
all p > 0.3), or in the summer group (−0.76 ± 2.71, −1.35 ± 2.71, −3.38 ± 2.82 for the 1, 4, and
6.5 PMB doses, respectively) compared to the placebo group (0.90 ± 1.99 nmol/L, all p >
0.2).
Cortisol in hair: This was not included in the overall health composite score as it was
only assessed at baseline and after 4 weeks. For three participants (1 in 1 PBM, 1 in 4 PBM
and 1 in 6.5 PBM) it was not possible to quantify cortisol in hair.
A significant increase over time in the amount of cortisol in the participant’s hair was
observed for the 1 J·cm–2 PBM condition (2.19 ± 0.87 pg/mg, p < 0.05) compared to placebo
(−0.75 ± 0.24 pg/mg). The analysis of the season factor revealed no effect of the PBM treat-
ment at any dose on the changes of the accumulated cortisol concentration in the winter
group (1.05 ± 1.21, 0.07 ± 1.21, −0.07 ± 1.21 for the PBM 1, PBM 4, and PBM 6.5 conditions,
respectively) compared to the placebo (−0.16 ± 0.86 pg/mg, all p values > 0.4). However,
the summer group showed a significant increase of accumulated cortisol levels in 1 J·cm–
2 PBM dose (3.56 ± 1.32 pg/mg, p < 0.05) compared to placebo (−1.57 ± 1.01 pg/mg).
Biology 2023, 12, 60 14 of 24
Figure 5. Overview of all individual elements of the health composite score for the summer and
winter separately. (A) IFN-γ, (B) TNF-α, (C) cortisol 0.5 h before bedtime, and (D) RHR of the night
after 2 and 4 weeks of PBM. Significance codes: ** p < 0.01, * p < 0.05, # p < 0.1 ns: not significant.
Sample sizes per condition are shown.
Resting heart rate (RHR): Resting heart rate was assessed continuously using a wear-
able sensor throughout the period of the study. To include this parameter in the health
composite score, the RHR assessed at night after 2 and 4 weeks was compared to the RHR
at the baseline night in a similar way as with the previous parameters. For two participants
(i.e., 1 in in the placebo group and 1 in in 6.5 PBM treatment group), it was not possible to
measure RHR, and one participant in the 4 PBM group did not wear the device. No inter-
action effect was observed between the PBM dose and time (F3,45 = 1.54, p = 0.22). The
cumulative average over 2 and 4 weeks showed no significant effects of PBM treatment
(−0.66 ± 1.39, −0.79 ± 1.39, −2.35 ± 1.41, for 1, 4, and 6.5 PBM, respectively) compared to the
placebo (1.03 ± 1.02, all p > 0.1). When accounting for the season, the analysis showed that
the RHR was significantly reduced in the winter group with 6.5 PBM treatments (−4.60 ±
1.90 bpm, p < 0.05) compared to the placebo (1.85 ± 1.34 bpm) but only a tendency in the
summer group (0.45 ± 2.15 bpm versus placebo −0.11 ± 1.59 bpm, p = 0.08). See Figure 5D,
Table S2C.
Analyzing the daily pattern of the RHR over the whole four-week period, it was
noted that the changes in the RHR were significantly lower on days that the PBM module
was used in the 4 PBM group (−2.73 ± 0.99 bpm, p < 0.01) compared to the placebo (64.26
± 0.74 bpm). Further analyses of the seasonal factor showed that these effects were signif-
icant in the winter group for both the 4 PBM (−5.7 ± 1.61 bpm, p < 0.001) and the 6.5 PBM
dose (−4.1 ± 1.65 bpm, p < 0.05) compared to the placebo group (66.5 ± 1.19 bpm). A signif-
icant interaction effect with season was observed for the 4 PBM dose (p < 0.05) and only a
Biology 2023, 12, 60 15 of 24
tendency with the 6.5 PBM one (p < 0.1), indicating that the effects were not present in the
summer group (Figure 6A,B). The factor days was not significant (p = 0.7) meaning that
RHR remained at a given level throughout the four weeks. The lowering of RHR appeared
to be maintained on days that the PBM module was not used PBM use (y/n) was not sig-
nificant (p = 0.8) nor was its interaction with the PBM dose (all p > 0.2).
Figure 6. Daily RHR during the four week period. The baseline values are shown on the left side.
The black circles represent the 0 J·cm−2 condition (n = 7:6 for winter and summer), grey squares the
1 J·cm−2 condition (n = 7:7 for winter and summer), pink triangles the 4 J·cm–2 condition (n = 8:7 for
winter and summer), and red triangles the 6 J·cm–2 condition (n = 8:6 for winter and summer). (A)
shows the data in the winter group and (B) of the summer group.
On a shorter time-frame (i.e., day 1 and day 2) the PBM dose did not reveal a signif-
icant interaction with time (F3,44 = 1.97, p = 0.13). The cumulative average over 1 and 2 days
showed a significant reduction of treatment on the RHR in both the 4 PBM group (−1.79 ±
0.6 bpm, p < 0.01) and 6.5 PBM group (−1.84 ± 0.61 bpm, p < 0.01) compared to the placebo
(0.76 ± 0.45 bpm). Consistent with other results, significant changes in RHR were only
present in the winter group in the 4 PBM (−2.26 ± 0.8 bpm, p < 0.01) and the 6.5 PBM group
(−2.57 ± 0.83 bpm, p < 0.01) compared to the placebo group (1.47 ± 0.61 bpm) (Figure 7). A
trend for a reduction was also observed in the one PBM condition (−1.61 ± 0.83 bpm, p =
0.06). No significant effects of PBM treatment were observed in the summer group (−0.63
± 0.87, −1.27 ± 0.90, −0.96 ± 0.9, for the 1, 4, and 6.5 doses, respectively) compared to the
placebo group (−0.09 ± 0.67 bpm, all p > 0.2). For four participants (i.e., 2 in placebo, 1 in 4
PBM and 1 in 6.5 PBM) RHR data were not available for one of the nights, and therefore
they were excluded from the analysis.
Figure 7. Short-term effects of PBM treatment (average between one stimulation and baseline and
two stimulations and baseline) on resting heart rate for summer and winter separately. Significance
codes: ** p < 0.01, # p < 0.1, ns: not significant.
Biology 2023, 12, 60 16 of 24
3.3. Effects of PBM Treatment on Sleep Quality
The analysis of the effect of PBM treatment on the composite score of sleep quality
revealed no significant interaction between the PBM dose and time (F3,48 = 1.53, p = 0.22).
The cumulative average over 2 and 4 weeks showed no significant effect of PBM treatment
at any dose on sleep quality compared to the placebo group (all p > 0.1). The analysis of
the season factor revealed no further effects of the PBM treatment, neither in the winter
nor in the summer group (Figure 3C, Table S2, Table S3A).
3.4. Effects of PBM Treatment on Circadian Rhythm
Dim Light Melatonin Onset (DLMO: PBM treatment on DLMO revealed no signifi-
cant interaction between time and dose (F3,33 = 0.83, p = 0.46). The cumulative average over
2 and 4 weeks showed no significant changes in the phase of the melatonin rhythms fol-
lowing PBM treatment for any dose (0.08 ± 0.37, −0.00 ± 0.39, 0.46 ± 0.39, for 1, 4, and 6.5
PBM doses, respectively) compared to the placebo (−0.48 ± 0.28, all p > 0.2).
In view of the number of participants for which the DLMO was not possible to cal-
culate, the analysis of the season factor was not possible. A total sample size of 9, 12, 10,
and 10 were available for the placebo, 1, 4, and 6.5 PBM, respectively. Out of the nine
available in the placebo group, only three belong to the summer group.
On a shorter time-frame (i.e., day 1 and day 2), the PBM dose showed no significant
interaction with time (F3,36 = 1.21, p = 0.32). The cumulative average over 1 and 2 days
showed no significant effects of PBM treatment compared to the placebo group (-all p >
0.6). In view of the limited number of participants for whom it was possible to analyze
DLMO, the analysis of the season factor is not possible. A total sample size of 8, 11, 13,
and 12 were available for the placebo, 1, 4, and 6.5 PBM, respectively. Out of the eight
available in the placebo group, only three belong to the summer group.
aMTs6: For five participants (i.e., 1 placebo, 1 in in 1 PBM group, 1 in in 4 PBM group
and 1 in in 6.5 PBM group) it was not possible to quantify aMTs6. Further, one participant
(6.5 PBM) did not collect night-time urine during the study. No significant interaction be-
tween the PBM dose and time (F3,42 = 0.46, p = 0.71) was observed. There was no indication
of a change in the amount of melatonin degradation product produced at night following
the PBM treatment compared to the placebo group (all p > 0.6). The analysis of the season
factor revealed no further effects of the PBM treatment, neither in the winter nor in the
summer group, see Supplementary Materials Table S3.
On a shorter time-frame (i.e., day 1 and day 2), PBM treatment did not result in a
significant interaction effect with the PBM dose and time (F3,44 = 1.35, p = 0.27). The cumu-
lative average over 1 and 2 days showed no significant effects of PBM treatment on aMTs6
compared to the placebo group (all p > 0.4). The analysis of the season factor revealed no
further effects of the PBM treatment, neither in the winter nor in the summer group, see
Table S4. For the short time-frame, three participants (i.e., 1 in placebo, 1 in 4 PBM and 1
in 6.5 PBM) it was not possible to quantify aMTs6. Further, one participant (6.5 PBM) did
not collect night-time urine during the study.
3.5. Temperature
During PBM treatment, no significant changes in skin temperature for the 4 and 6.5
PBM groups compared to the placebo were found, irrespective of anatomical location (all
p > 0.2). This indicates that there was no acute effect on the temperature or any indications
for thermoregulatory process from the PBM treatments. For the one PBM dose, a signifi-
cant increase in temperature (0.82 °C) at the head location only was observed (t = 2.64, p <
0.05, Figure S6).
4. Discussion
The goal of the present study was to assess whether a PBM set-up used at home with
a treatment period of several hours per day during several weeks could be beneficial for
Biology 2023, 12, 60 17 of 24
general well-being, health, sleep quality, and circadian entrainment in healthy subjects
with mild sleep-related complaints. The analysis of the composite scores showed that
well-being and health were positively affected at the highest dose of PBM (6.5 J·cm−2) dur-
ing winter, while estimates of sleep quality were not affected. Nor were any circadian-
related outputs affected by PBM treatment. To our knowledge, this is the first time that
the systemic effects of PBM have been studied in healthy subjects during their normal
daily routine in a double-blind, randomized, placebo-controlled trial.
Near-infrared (NIR) stimulation is a natural phenomenon that occurs when humans
are exposed to sunlight. The positive health effects of sunlight have often been attributed
to the vitamin-D3, -endorphin, serotonin production in response to UV-B exposure [47–
49]. In a recent paper, Heiskanen et al. questioned the role of the effects of vitamin D3
alone and suggested that red and NIR light may also be a component of the positive effects
on health [6]. It should be noted that indoor NIR irradiance from general lighting condi-
tions is at least 100 times lower than direct sunlight. This is likely too low to induce an
appreciable biological benefit, even with previously used incandescent light sources,
which do emit NIR radiation. Achieving 500 lux using 3000 K incandescent lamps delivers
an irradiance of 1.5 W·m−2 in the 800−900 nm spectral window. Even if the low irradiance
of 1.5 W·m−2 would be sufficient to reach the PBM threshold, it would require almost 12 h
exposure to achieve a 6.5 J·cm−2 PBM dose.
The 6.5 J·cm−2 PBM dose is a reasonable treatment similar to natural sunlight expo-
sure. If people were outside and uncovered on a clear summer’s day in The Netherlands,
we could expect an 800−900 nm irradiance of about 90 W.m−2 at midday [50]. The 6.5 J·cm−2
PBM condition used in the present study would then be achieved after about 12 min of
natural light exposure. On a clear winter’s day, this would be achieved after about 16 min,
while on a cloudy day one would need to spend about 7 h outdoors (energy of about 2.5
W·m−2 at midday for the 6.5 J·cm−2 PBM condition [50]. Considering that in the winter
months people wear protective clothes with very little skin exposed to sunlight due to the
weather conditions, and that sunny winter days are exceptional occasions in Northern
Europe, the 6.5 J·cm−2 NIR exposure would rarely ever be reached. The ease with which a
natural PBM dose can be experienced in summer might explain the consistency of our
stand-out findings occurring only in the winter group, even though the sample size for
the season comparison was relatively small and should be considered a limitation on
which to improve in following studies.
In the present study, many parameters were analyzed. To gain statistical power,
three main composite scores were created to test for overall effects on well-being (several
subjective ratings), health (several physiological markers), and sleep (several subjective
and objective estimates of the sleep-wake rhythm). A more detailed analysis of the well-
being and health composite scores showed consistent results for various parameters. In
the well-being category, drowsiness and mood mostly contributed to the positive PBM
effects (Figure 4). This is strengthened by the consistent finding that mood was already
improved by the highest PBM dose immediately after the first day and for the following
four weeks. PBM, mostly transcranially, has repeatedly been shown to be able to posi-
tively influence mood in different sorts of conditions (e.g., brain injury, traumatic events,
and depression, as well as in healthy subjects) [51]. PBM has also recently been discussed
as a tool for the treatment of depression, but the authors do not outline optimal treatment
parameters as the variation in these reported parameters is too large to be summarized
[18,52]. Using the BDI as a scale, Henderson et al. found, unlike us, a reduction in de-
pressed mood [53]. The study included participants who had a much higher BDI score at
inclusion about 24 compared to our baseline value of about 11, and much higher NIR ir-
radiances were used (13.2 W at 0.89 cm2, 810 nm). We explicitly excluded potential candi-
dates with a BDI above 19, which has been linked to moderate depression [40], as we
wanted to test the PBM intervention in healthy subjects. In addition, we aimed at identi-
fying an effect with the lowest possible intensity. Nonetheless, we saw a trend for a re-
duction in depression in the 6.5 J·cm−2 PBM condition during the winter months (Figure
Biology 2023, 12, 60 18 of 24
S5). This interaction between PBM and the season was also observed for the well-being
composite score. This could be explained by the fact that the participants were more likely
to be exposed to natural sunlight during the summer months to such an extent that the
additional PBM treatment had no effect on the subjective parameters contributing to the
well-being composite score. To some extent, the increase in vitamin D levels in the sum-
mer group over the four weeks in all conditions confirms this. The positive effects on well-
being being visible in winter, and significantly different from the placebo group strength-
ens the conclusion that it is the 6.5 J·cm−2 PBM treatment that is responsible for the positive
effects. Furthermore, the well-known seasonal variation that some of the well-being items
show, by which a decrement is observed in the winter months [54], might have contrib-
uted to the observed interaction, i.e., more room for improvement during the winter
months.
The composite score for health also showed a positive effect in the 6.5 J·cm−2 PBM
group. This is primarily due to a reduction in cytokine IFN-γ and a reduction in the resting
heart rate (Figure 6). IFN-γ is key in driving inflammatory responses against both exoge-
nous and endogenous species [55,56]. Downregulation of IFN-γ after exposure to far-red
(670 nm) light has already been shown in mice after full body exposure [57] and in vitro
[58]. In general, one of the most reproducible effects of PBM is a reduction in inflammation
[59], but to our knowledge, this is the first study that shows such a systemic reduction in
humans after a four-week treatment period. The positive effect of PBM in reducing in-
flammation has even resulted in a discussion as a potential treatment for COVID-19 [60–
62]. An anecdotal additional finding to this discussion is that during the current study
carried out in the year 2021, only 1 out of the 62 initially included participants (PBM con-
dition 4 J·cm−2) tested positive during the four-week study. This participant showed only
mild symptoms and recovered within two days. With a very high rate of infections this
year, this is an intriguing finding. Future studies, testing specifically on markers for
COVID-19, could provide further support for a possible protective role of PBM on the
immune system. The cytokine TNF-α was significantly reduced in the 6.5 J·cm−2 dose
group only when BMI was considered as part of the model (Table S5). The interaction
effect indicates that a higher BMI hampers to some extend the reduction of TNF- by PBM.
This effect could be explained by the different metabolism of fat cells in obese people com-
pared to non-obese people. The production of cytokines amplifies with increasing BMI
[63,64] and fat cells of obese people produce 5 to 10-fold higher TNF- mRNA compared
to lean fat cells [65,66]. It may be interesting to test people who are overweight and show
high levels of inflammation for a longer period than 4 weeks and/or with higher doses of
NIR.
The selection of this particular group of subjects was premised on the mitochondrial
mechanism of PBM. This is the most accepted mechanism of action, and it is based on
photons being absorbed by cytochromes, which are present inside the mitochondria either
as functional proteins or as electron transport shuttles (i.e., cytochrome c oxidase, CCO).
This leads to an increased production of ATP, increased oxygen consumption, raised mi-
tochondrial membrane potential, and increased mitochondrial biogenesis, which have all
been shown in vitro after PBM, resulting in transient increase in ATP, ROS, and NO levels
[9,20,21,59,67]. Mitochondrial dysfunction is thought to be a causal factor in the detri-
mental health effects of sleep deficits, especially in people who are late chronotypes
[30,31]. The reported potential role of NO having a positive effect on mood may be rele-
vant as well. The positive effects of ambulatory PBM treatment during daytime hours, if
effective via mitochondrial stimulation, may be particularly suitable for the general public
suffering from sleeping problems. Although the positive health and well-being effects
were indeed noted in the current study, they do not seem to be mediated by positive ef-
fects on the circadian rhythm or sleep. Further insight into the mechanisms underlying
the effects is necessary. It is feasible that PBM may modulate immune cell functions by
modulating their mitochondrial functions. This explanation has been hypothesized to be
one of the mechanisms by which PBM could assert a systemic effect [68]. The positive
Biology 2023, 12, 60 19 of 24
effects of light on the skin and the role of humoral phototransduction in the effects on
mood have been discussed in other contexts, such as light exposure during winter depres-
sion [69,70]. However, studies on light treatment at visual wavelengths through the skin
were not found to be effective in treating mood or affecting the circadian system [71]. The
evidence for direct modulation of the cytokines IFN- and TNF-α levels in this study raises
interesting possibilities of other systemic factors that are being directly modulated by
PBM treatments, as has been shown with TGF-1 [72]. Moreover, several studies report
that pro-inflammatory cytokines play an important role in the brain in the pathogenesis
of mood disorders [73–75]. This study noted a significant reduction in cytokines levels
and improved mood which may be an interesting aspect of future PBM studies, poten-
tially providing mechanistic insights for therapeutic PBM responses.
Another parameter contributing to the composite score ‘health’ is the resting heart
rate (RHR). The RHR represents the balance of sympathetic and parasympathetic activity
and is considered a reliable marker of autonomic nervous system tone [76]. The associa-
tion between an increased RHR and adverse health outcomes in the general population
has been widely investigated [77]. We observed an immediate change in the RHR after the
first PBM session (4 J·cm−2 and 6.5 J·cm−2 PBM conditions), that is maintained throughout
the whole study (Figure 7). We included cortisol levels at bedtime as a fourth item in the
composite score for health. Higher evening cortisol levels have been found in mood dis-
order patients [78–80] and may be related to higher stress and arousal levels. Higher bed-
time levels of cortisol have also been reported to be related to disrupted sleep [81,82].
Although a reduction in cortisol at bedtime was observed after treatment with 6.5 J·cm−2,
this was no longer visible when exploring the differences between the winter and summer
groups. This could be attributed to lower statistical power. Cortisol earlier in the evening
was not affected by PBM, suggesting that the effects are indeed more related to the ease
of the moment just prior to falling asleep. The lack of a consistent effect of PBM on the
accumulated cortisol level over 4 weeks as measured in hair samples does not support the
idea that PBM reduces cortisol in general.
Although we did not specifically seek to identify side effects in this study, from our
regular contact with the participants, a few negative experiences were exchanged. These
included headaches, eyestrain, dizziness, tiredness, and dryness of the skin (Table S6). As
evident from the data, these reports constituted a very small number of participants (be-
tween 1 and 3) and did not correlate with the PBM dose specifically, suggesting that the
way PBM treatment was delivered was largely acceptable.
5. Conclusion
The findings from the current study, that 6.5 J·cm−2 PBM treatment improves several
health and well-being-related factors, are supported by previous reports on the beneficial
effects of PBM. That our findings are consistent in the short and long term and only pre-
sent in the winter strengthens our observations. Still, a replication of the present study in
terms of its set-up (i.e., home, LED-based) is desired, as well as studies on possible cellular
mechanisms and pathways involved in mediating the effects on health and well-being.
Future prospective research on dose, duration, timing, and potential mechanisms is ex-
pected to further strengthen our conclusions.
The present study only investigated rather a low-energy PBM treatment, and the op-
timal dosage might not have been reached. While it is known that increasing energy could
intensify the stimulation and the possible effects before reaching a plateau [15], the present
positive findings at such low intensities suggest the possibility of the incorporation of
PBM into household or personal appliances (i.e., considering energy costs). It seems im-
portant to intensify research on the effects of non-visual long wavelengths in addition to
the important research on the effects of visual light on non-image-forming functions
[83,84]. In light of our indoor lifestyle and the need for more healthy buildings, the current
results may open a completely new way of creating an optimal environment for a health-
ier society.
Biology 2023, 12, 60 20 of 24
6. Patents
The patent number of NIR-LEDs is WO2020119965A1, can be found at https://pa-
tents.google.com/patent/WO2020119965A1/en?oq=wo+2020%2F119965A1 (accessed on 7
November 2022).
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/biology12010060/s1, Table S1: Demographics summer and
winter; Table S2: Linear model long-term effects overview; Table S3: Linear model no long-term
effects overview; Table S4: Linear model short-term effects overview; Table S5: Linear model includ-
ing BMI interaction overview; Figure S1: Raw data well-being; Figure S2: Raw data health; Figure
S3: Raw data sleep; Figure S4: Ambient light and vitamin D; Figure S5: Depression scores; Figure
S6: Skin temperature.
Author Contributions: conceptualization, M.C.G., M.C.M.G., P.P.B.; methodology, M.C.G.,
M.C.M.G.; formal analysis, M.C.G., M.L., E.G.V.S., M.A.K., R.L.H., R.A.H., P.P.B.; investigation,
M.C.G., M.C.M.G., P.P.B., M.L., E.G.V.S., M.A.K.; data curation, M.C.G., M.C, M.L., E.G.V.S.,
M.A.K., R.L.H.; writing—original draft, M.C.G., M.C.M.G.; writing—review and editing, M.C.G.,
M.L., E.G.V.S., M.A.K., R.L.H., P.R.A., A.C.B., M.R.K., R.A.H.; visualization, M.C.G.; supervision
M.C.G., M.C.M.G., M.L., P.P.B.; project administration, M.C.G., M.C.M.G., P.P.B., M.L.; funding ac-
quisition, M.C.M.G., M.C.G. All authors have read and agreed to the published version of the man-
uscript.
Funding: This research was funded by Seaborough Life Science B.V. Agreement 25092020.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Ethics Committee of the University Medical Center in
Groningen (protocol code NL74857.042.20 approved on 16 November 2020)
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: Not applicable.
Acknowledgments: The authors want to thank Jürgen Honold and Charlie Minter for their help in
the preparation of this clinical study, and the electronics Department of Seaborough Research BV
for their efforts developing the PBM test module. We would also like to thank N.M. Jansonius, from
the faculty of medical sciences at the University Medical Center in Groningen for his role as an
independent expert. We further acknowledge D. Kalsbeek (University of Amsterdam, NIN), Ancora
Health B.V., DC Klinieken, CERTE Groningen, and the University Medical Center in Groningen for
facilitating blood samples withdrawal and storage.
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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