Hartstein et al.
Journal of Physiological Anthropology(2024) 43:16
https://doi.org/10.1186/s40101-024-00363-6

Journal of
Physiological Anthropology

ORIGINAL ARTICLE

Open Access

Differences in the pupillary responses
to evening light between children
and adolescents
Lauren E. Hartstein1* , Monique K. LeBourgeois1, Mark T. Durniak2 and Raymond P. Najjar3,4,5,6

Abstract
Background In the mammalian retina, intrinsically-photosensitive retinal ganglion cells (ipRGC) detect lightand integrate signals from rods and cones to drive multiple non-visual functions including circadian entrainment
and the pupillary light response (PLR). Non-visual photoreception and consequently non-visual sensitivity to light
may change across child development. The PLR represents a quick and reliable method for examining non-visual
responses to light in children. The purpose of this study was to assess differences in the PLRs to blue and red stimuli,
measured one hour prior to bedtime, between children and adolescents.
Methods Forty healthy participants (8–9 years, n = 21; 15–16 years, n = 19) completed a PLR assessment 1 h
before their habitual bedtime. After a 1 h dim-light adaptation period (< 1 lx), baseline pupil diameter was measured in darkness for 30 s, followed by a 10 s exposure to 3.0 × ­1013 photons/cm2/s of either red (627 nm) or blue
(459 nm) light, and a 40 s recovery in darkness to assess pupillary re-dilation. Subsequently, participants underwent
7 min of dim-light re-adaptation followed by an exposure to the other light condition. Lights were counterbalanced
across participants.
Results Across both age groups, maximum pupil constriction was significantly greater (p < 0.001, ηp2 = 0.48)
and more sustained (p < 0.001, ηp2 = 0.41) during exposure to blue compared to red light. For adolescents, the postillumination pupillary response (PIPR), a hallmark of melanopsin function, was larger after blue compared with red
light (p = 0.02, d = 0.60). This difference was not observed in children. Across light exposures, children had larger phasic(p < 0.01, ηp2 = 0.20) and maximal (p < 0.01, ηp2 = 0.22) pupil constrictions compared to adolescents.
Conclusions Blue light elicited a greater and more sustained pupillary response than red light in children and adolescents. However, the overall amplitude of the rod/cone-driven phasic response was greater in children than in adolescents. Our findings using the PLR highlight a higher sensitivity to evening light in children compared to adolescents,
and continued maturation of the human non-visual photoreception/system throughout development.
Keywords Children, Adolescents, Pupillary light reflex, Light exposure, Intrinsically photosensitive retinal ganglion
cells

Dr. LeBourgeois passed away in November 2023.
*Correspondence:
Lauren E. Hartstein
Lauren.Hartstein@colorado.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

Background
Intrinsically photosensitive retinal ganglion cells (ipRGCs)
are ocular photoreceptors that express the photopigment
melanopsin and are maximally sensitive to short-wavelength blue light, with a peak sensitivity of ~ 480 nm [1,2]. ipRGCs integrate their intrinsic response to inputs
from rods and cones, before transmitting the signal tonon-visual centers in the brain such as the suprachiasmatic nucleus (SCN), the central timekeeper of bodily
circadian rhythms [3, 4], the olivary pretectal nucleus(OPN) which controls the pupillary light response (PLR)[5, 6], and centers involved in sleep and alertness [7].
Stimulation of ipRGCs by light in the evening can suppress the production and release of the sleep-promoting
hormone melatonin from the pineal gland, delay the timing of the circadian clock, and decrease nighttime sleepiness [8, 9]. These effects may be particularly strong inchildren, compared with adolescents or adults [10–12].
The PLR offers a fast and reliable biomarker for testing
the physiology and integrity of visual and non-visual photoreception. It generally consists of a rapid constrictionof the pupil to an abrupt light stimulus. This transient(or phasic) constriction response is largely attributed toouter-retinal (rod/cone) activity [6]. If the light stimulation is maintained, the transient constriction is followed
by a sustained constriction of the pupil (steady-state).
The steady-state response consists of input from all photoreceptors; however, the ipRGC’s intrinsic contribution
to this state is three-folds higher than long-wavelength(L-cones) and medium-wavelength (M-cones) cones [5].
After cessation of the light stimulus, signals from both
the rods and ipRGCs contribute to the post-illumination pupil response (PIPR) in the first 1.7 s, after which
the PIPR is entirely driven by ipRGCs [13]. Chromatic
pupillometry, using light stimuli of various colors/wavelengths, is increasingly used to measure retinal healthand development [14, 15]. By employing light exposure
patterns of different wavelengths, this technique has beensuggested to differentiate pupillometric signals originating from the outer (rods and cones) and inner (intrinsic ipRGC response) retina according to the irradianceand spectral sensitivities of photoreceptors. In adults,
exposure to short-wavelength blue light results in faster,
greater, and more sustained pupillary constriction thanlong-wavelength red light [16–18]. This more sustained
response to blue light is consistent with greater stimulation of the ipRGCs. Therefore, components of the chromatic PLR can be used as an indicator of the ipRGC
response and can be utilized as a marker in health anddisease [15, 19, 20], even in clinical settings [21].
Baseline pupil diameter increases throughout childhood, peaking in adolescence [22–24], then slowly

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decreases throughout adulthood [10, 25, 26]. In adults
age 21–70 years, a larger baseline pupil diameter was
associated with a larger PIPR 6 s after exposure to blue
light [25]. However, when controlling for baseline pupil
diameter, age was not associated with PIPR, indicatingthat the inputs of the ipRGC to the pupillary pathway
do not diminish with advancing age. Data on the maturation of the PLR throughout child development, however, remain limited. In premature infants (gestationalage 38 weeks), the PLR is observed in response to blue
but not red light [24]. However, the magnitude of thepupillary constriction, as well as baseline pupil size, issignificantly smaller in premature infants than at 2 yearsof age [24]. In a longitudinal study of the PLR elicited bygreen light (530 nm) across children aged 6–24 months,
baseline size, minimal pupil size, and maximum pupil
constriction (% constriction with respect to baseline)
increased with age, whereas latency (the time fromlight onset to the start of pupil constriction) decreased
with age [27]. In a sample of children aged 6–17 years,
the amplitude of pupillary constriction in response to
green light increased until age 8, then plateaued [23].
PLR latency decreased across ages 6–9, then stabilized.
Constriction and re-dilation time were not associatedwith age. Across children ages 5–15 years, the ipRGCdriven pupil response to light was observed to be robust
and similar to those measured in adults [28]. Lastly,in another study examining the PLR between children
aged 3–10 and 10–18 years, the younger group requireda brighter red light stimulus to evoke a 5% pupil constriction than the older group [14].
The circadian timing of a light exposure can influence
the PLR [29]. In adults, circadian fluctuations in poststimulus pupil size were observed in response to blue
(463 nm) but not red (635 nm) light [30]. Additionally,
the ipRGC-driven pupil response in children is influenced by individual differences in 24-h light history [28].
Furthermore, across the lifespan, humans spend the vast
majority of their time in indoor environments [31, 32],
resulting in less exposure to bright light during the day
and greater exposure to artificial light at night [33]. Boththe circadian timing of assessments and prior light history are largely unreported and uncontrolled for in previous studies of the PLR in children. The purpose of thepresent research was to (1) study the chromatic pupillary
light reflex in response to red and blue evening light inchildren and adolescents while controlling for the confounding factors mentioned above; and (2) examine
age-related differences across 2 distinct stages of childdevelopment. The measurement was conducted in thehour before participants’ habitual bedtimes in order
to simulate conditions under which children are often

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

exposed to artificial light and a time when the non-visualcircadian system is highly responsive to light exposure.

Methods
ParticipantsChildren aged 8.0–9.9 years (n = 21, 12 female, 81.0%
white, 4.8% Asian, 14.3% more than one race reported,
90.5% non-Hispanic, M = 8.99 years, SD = 0.51 years) andadolescents aged 15.0–16.9 years (n = 19, 5 female, 94.7%
white, 5.3% more than one race reported, 100% nonHispanic, M = 15.78 years, SD = 0.55 years) completed a
6-day study protocol. Participants were recruited fromthe greater Boulder, CO, USA area. Prospective interested parents were screened through an online questionnaire and subsequent phone interview. Participants
were excluded from the study for parent report of any of
the following: clinical sleep disorders; behavioral problems; personal or family history of diagnosed narcolepsy,
depression, psychosis, or bipolar disorder; pre-termdelivery (< 35 weeks); low birth weight (< 5.5 lbs.); current
use of caffeine, nicotine, alcohol, or medications affecting sleep, the circadian system, or light sensitivity; active
allergies or asthma; developmental disabilities; neurological disorders; metabolic disorders; chronic medical
conditions; head injury involving loss of consciousness;travel > 2 time zones in the 2 months before participation; sleep schedule varying > 2 h between weekdays andweekends; migraine or frequent headaches; eye disorders (excluding corrected refractive error) or color blindness (confirmed using the Ishihara Color Vision Test).
All study procedures were approved by the University ofColorado Boulder Institutional Review Board. Parents
provided written, informed consent and children/adolescents provided written assent to participate. Families
were compensated for their participation.

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ProtocolData collection occurred between June and Octoberin 2021 and September through October in 2022. For
5 days, children followed a strict parent-selected sleep
schedule (bedtime and wake time) with ≥ 9 h time in bed,
whereas adolescents followed a self-selected sleep schedule of ≥ 8 h time in bed [34]. Adherence to the sleep
schedule was confirmed through a wrist-worn actigraph(Spectrum Plus, Philips Respironics, Pittsburgh, PA,USA) which recorded sleep–wake states via motor activity in 1 min epochs, as well as a daily sleep diary (parentreported for the school-aged children and self-reported
for the adolescents). Participants were instructed to
avoid light exposure between bedtime and wake. Starting at wake time on Day 6, participants wore a pair ofdark glasses (UVEX by Honeywell, Espresso lens) with anadvertised visible light transmission of 15% [35] in order
to limit exposure to bright light and reduce individual
variability in light history (Fig. S1). The transmittance ofthe glasses at 480 nm was measured as 8.9%. Participants
were instructed to remain indoors wearing the glasses
throughout the day until beginning the dim-light adaptation at the laboratory that evening (Fig. 1). Participants
arrived at the lab 2.5 h before their scheduled bedtime forthe pupillary assessment. They then entered a dimly-lit
room (< 1 lx at angle of gaze) beginning 2 h before theirscheduled bedtime and adapted to the dim-light for 1 h.
The pupillary assessment then began 1 h before eachparticipant’s scheduled bedtime. Researchers fitted
the participant with a head-mounted eye tracking unit
(ETL-100F, ISCAN, Inc., Woburn, MA, USA), whichmeasures pupil diameter at a rate of 60 Hz. Participants
were seated at a table and instructed to keep their chinon a chinrest ~ 42 cm from the light source and lookstraight ahead during all measurements. Pupil diameter

Fig. 1 Pupillary assessment protocol. On the day of the pupillary assessment, participants remained indoors wearing a pair of dark-tinted glasses
from their habitual wake time until the start of the dim-light adaptation at the laboratory (2 h before scheduled bedtime). Following a 1 h dim-light
adaptation, pupil diameter was measured during a 30 s baseline, 10 s light exposure, and 40 s recovery. After a 7 min dim-light re-adaptation,
the measurement was repeated for the remaining light condition, counterbalanced across participants.

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

was recorded during a 30 s baseline in darkness, a 10 slight stimulus, and a 40 s recovery period in darkness tomeasure pupillary re-dilation. Participants were exposed
to either a blue or red narrow-band LED light source
matched in photon flux at 3.0 × ­1013 photons/cm2/s(Table 1; Fig. S2). The light source consisted of LED strips(YUNBO, Changzhou, China) arranged on a backplane ofa 61 cm × 61 cm × 12 cm deep white box with an acrylic
diffusing panel at the front. A research spectrometer(MSC15, Gigahertz-Optik, Amesbury, MA, USA; calibrated 04/01/2021) was used to verify light intensity on
the vertical plane from the chinrest before each participant. Following a 7 min re-adaptation to the dim-light,
the measurement was repeated for the remaining lighting
condition, the order of which was counterbalanced acrossparticipants. For four participants (three children, one
adolescent), a visual scan of the data revealed an error in
the recording. These children were re-adapted to the dim
light for an additional 7 min before the measurement was
repeated. Light metrics are reported in accordance with
the ENLIGHT Checklist and Guidelines [36].
Analysis

Actigraphy data were scored using our standard, published methods [37]. Light exposure at the wrist wasrecorded by the actigraph in 1 min epochs. Sleep

Table 1 Spectral characteristics for each experimental lightsourceLight parameter

Blue

Red

Peak wavelength (nm)459.08 (0.36)

627.12 (0.29)

Full-width half maximum (nm)24.56 (0.15)

20.67 (0.13)

Photopic illuminance (lux)7.17 (0.24)

22.15 (0.67)

Melanopic equivalent daylightilluminance (lux)70.09 (2.32)

0.10 (0.01)

Measurements were obtained with a research spectrometer before eachparticipant’s assessment. Means (standard deviations) across all participants arepresented in the table above

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parameters (sleep duration, sleep efficiency, wake after
sleep onset (WASO)) were calculated by averaging acrossthe five nights prior to the pupillary measurement. Lighthistory across the first five days of the protocol was calculated as the average across each epoch between 00:00
on Day 1 and 23:59 on Day 5 of the protocol. Light history on Day 6 was calculated as the average across epochs
between 00:00 and each participant’s arrival at the lab for
their pupillary assessment (2.5 h before their scheduled
bedtime), at which time the actigraph was removed andeach participant was exposed to the same lighting environment in the lab prior to the PLR measurement.
Pupil diameter data were analyzed only from the righteye of each participant and recorded in raw pixels. Alldata processing was performed using Matlab R2021b(The MathWorks, Inc., Natick, MA, USA). Prior to
extracting variables, the data were filtered and smoothed
to remove blinks. This was achieved by first manually setting upper and lower thresholds to exclude outliers dueto blinks. Then, the data were smoothed using the Matlab ‘lowess’ smooth function with a span of 0.01. The PLRvariables (adapted from Najjar et al. 2023 [15]) were thenextracted from this smoothed data (Table 2).
First, a baseline pupil size, in pixels, was calculated as
the median diameter across the 30 s prior to light onset.
Each data set was then normalized to this baseline totranslate the pupil size in pixels to percent constriction
away from baseline, with 0% representing baseline pupilsize. Phasic constriction and constriction latency could
not be determined for two children due to a technicalerror in recording the exact moment of light onset during
one of their measurements.
All statistical analyses were conducted with SPSS Statistics 28.0 (IBM Corp. Armonk, NY, USA). Due to nonnormal distributions, Mann–Whitney U tests were used
to examine group differences in light history. A Wilcoxon
signed rank test was used to examine individual changes
in light history between Days 1 through 5 comparedwith Day 6. Independent samples t-test were utilized toTable 2 Description of each pupillary variable calculated
Variable Name (units)

Definition

Baseline Pupil Diameter (pixels)

Median pupil diameter in the 30 s preceding light onset

Phasic Constriction (% from baseline)

Median % constriction between 300–700 ms after light onset

Constriction Latency (s)Time required after light onset for the pupil to reach 10% of constriction

Maximum Pupil Constriction (% from baseline)

Diameter (during light on)
1 − Min Pupil
Baseline Pupil Diameter

Sustained Slope (%/s)

Slope of best fit linear regression from 3–10 s after light onset

Post-Illumination Pupil Response (PIPR, % from baseline)

Median percent constriction between 5 to 7 s after light offset

Area Under the Curve (AUC, %.s)

Absolute value of a trapezoidal numerical integral of the data
between light offset and 12 s after light offset

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

examine group differences in sleep parameters and baseline pupil diameter. PLR data were analyzed with mixedmodel ANOVAs to examine differences in response to
the two light conditions (Red, Blue) and across the agegroups (Children, Adolescents). Effect sizes for significant results are provided as partial-eta squared. Post-hoc
comparisons were performed with repeated measurest-tests, and effect sizes were determined using Cohen’s d.
An alpha-level of 0.05 was used to determine statisticalsignificance.

Results
Two participants were myopic, but did not wear corrective lenses during the pupillary assessments. Analyses were performed both including and excluding theirdata. As expected from the previous literature [38], we
observed no differences in the results of the analyses after
excluding these participants.

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researcher instructions to remain indoors until coming to
the laboratory in the evening.
Baseline pupil size

Baseline pupil diameter before each participant’s firstlight exposure was not significantly different between
children (M = 231.62 pixels, SD = 35.31) and adolescents(M = 242.88 pixels, SD = 29.23; t(38) = -1.09, p = 0.28).
Neither was any group difference observed in baseline pupil diameter before each participant’s second
light exposure (t(38) = -1.69, p = 0.10; M = 225.49 pixels,
SD = 34.42 for children; M = 242.18 pixels, SD = 27.31 foradolescents). A paired-samples t-test indicated a non-significant difference in baseline pupil diameter between thefirst and second measurements (t(39) = 1.85, p = 0.07).
Descriptive statistics for each PLR variable across lightcolor and age group are presented in Table S1.

Sleep parameters

Average sleep duration across the 5 nights preceding the in-lab assessment was significantly longer forthe children than the adolescents, commensurate with
reductions in sleep duration observed across typicaldevelopment (Table 3) [39]. However, no differences insleep efficiency or wake after sleep onset (WASO) wereobserved, indicating comparable sleep quality across thetwo age groups in the days leading up to the pupillarymeasurement.
Prior light exposure

Across the first five days of the protocol, average light
exposure as measured from the wrist was significantly
greater for children (M = 248.93 lx, SD = 80.88) than adolescents (M = 162.08 lx, SD = 147.94; z = -2.99, p < 0.01,
r = 0.47). On the day of the pupillary assessment (Day 6),
however, when participants were instructed to remainindoors throughout the day, no difference in light exposure between the two age groups was observed (children: M = 91.23 lx, SD = 70.28, adolescents: M = 64.82 lx,
SD = 52.20; z = -1.26, p = 0.22). Additionally, across individual participants, average light exposure on Day 6 wassignificantly less than on Days 1 through 5 (z = -4.80,
p < 0.001, r = 0.76), suggesting participants adhered to

Features of pupillary light constriction

Detailed descriptions of how each variable was calculated
are provided in Table 2. Average pupil constriction (as a% away from baseline) was plotted for each age group andlight condition (Fig. 2). Across both age groups, phasic
constriction was marginally greater in response to blue
than red light (p = 0.054, ηP2 = 0.10; Fig. 3A). Regardless of the wavelength of the stimulation, children had
both a significantly greater phasic constriction (p < 0.01,
ηP2 = 0.18) and smaller constriction latency (p = 0.01,
ηP2 = 0.17; Fig. 3B) than adolescents, indicating a fasterand more robust initial pupillary response to light in general. Maximum pupil constriction was greater in responseto blue light compared to red light for both age groups(p < 0.01, ηP2 = 0.48; Fig. 3C). Children also exhibited agreater maximum constriction than adolescents (p < 0.01,
ηP2 = 0.22). Between 3 to 10 s after light onset, pupil constriction was more sustained (i.e., a smaller magnitude
slope was observed) during blue compared to red light
across both age groups (p < 0.01, ηP2 = 0.41; Fig. 3D). Conversely, an independent samples t-test revealed a larger
magnitude slope during blue light in children than inadolescents (t(38) = 2.32, p = 0.03, d = 0.74), indicating aless sustained response.

Table 3 Actigraphically-derived average sleep duration and quality
VariableChildren

Adolescents

Statistics

Sleep Duration (min)578.71 (25.13)

504.21 (31.25)

t(38) = 8.35, p < 0.001

Sleep Efficiency (%)

85.28 (3.32)

84.66 (5.62)t(38) = 0.43, p = 0.67

Wake After Sleep Onset (min)63.34 (14.87)

62.34 (23.28)

t(38) = 0.16, p = 0.87

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

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Fig. 2 Average pupil constriction adjusted to baseline pupil diameter across the 80 s measurement. Average pupil constriction is displayed at eachtime point for (A) children and (B) adolescents for red and blue light separately. The shaded red and blue areas around each average line represent
the standard error of the mean. The shaded yellow area denotes the timing of the 10 s light exposure

Fig. 3 PLR metrics during light onset. Results depicting (A) phasic constriction; (B) constriction latency; (C) maximum pupil constriction; and (D)slope during light exposure broken down by age group and experimental light condition. Asterisks denote statistical significance (p < 0.05)

Features of pupillary re‑dilation

The area under the curve (AUC) 0–12 s after light offset
was significantly larger for blue compared to red light(p = 0.045, ηP2 = 0.10; Fig. 4A). However, post-hoc t-tests
revealed that this was observed for adolescents (p < 0.01,
d = 0.72), but not children (p = 0.66). Similarly, the PIPR
was larger following blue compared to red light in adolescents (p = 0.02, d = 0.60; Figure 4B), but not in children(p = 0.76). The PIPR following the red light was 3.92%
larger in children than adolescents, though this difference did not reach statistical significance (t(38) = 1.88,
p = 0.07). The PIPR following the blue light was 1.41%
larger for children than adolescents, indicating the lackof difference between light stimuli for the children was
the result of a more sustained response to red light rather
than a less sustained response to blue.

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

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Fig. 4 PLR metrics following light offset. Results depicting (A) area under the curve and (B) post-illumination pupil response (PIPR) broken
down by age group and experimental light condition. Asterisks denote statistical significance (p < 0.05)

Discussion
In this well-controlled, repeated-measures study examining age-related changes in the pupillary light reflex,
we observed a greater and more sustained pupillary
response to blue compared to red light presented in thehour before habitual bedtime for both school-aged children and adolescents. Additionally, although children
exhibited a faster and greater amplitude of transient andmaximal pupillary constriction across both light colors
compared to adolescents, they yielded a reduced sustained response to blue light.
Previously, Crippa and colleagues [14] reported thatchildren aged 3–10 years required a brighter red lightstimulus to reach 5% pupil constriction than children
10–18 years. Additionally, in children aged 6–17 years,
pupillary constriction in response to a green light stimulus has been shown to increase until age 8 before plateauing [23]. Similarly, latency was observed to decrease
across ages 6–9, then stabilized. In contrast, our findings suggest that maximum constriction to both red andblue light decreases and latency increases again between
school-age and adolescence. By examining more narrow age groups (8–9 and 15–16 years), our data offer amore fine-grained examination of age-related differencesin the PLR than prior published findings, with particularfocus around the onset of puberty where changes in the
non-visual response to light have been reported [12]. It isdifficult to compare findings across the few studies examining age-related changes in the PLR due to differencesin protocols and light stimuli, highlighting the need for
greater research in these populations and the development of well-controlled standardized procedures.

Our findings contribute to a growing literature demonstrating maturation of the non-visual system throughout development. Previous work by Crowley, et al. [12]demonstrated a reduced circadian response to light inlater adolescence. Pre-pubertal adolescents exhibited significantly more melatonin suppression to evening light
exposure than post-pubertal adolescents. Additionally,
school-aged children display nearly twice as much melatonin suppression to evening bright light as adults [10].
Our data are also in agreement with experimental findings in mice showing that the pupillary light reflex continues to develop until adulthood. In that study, the PLR in
response to blue and red light was compared in 1-monthold mice to 2-month-old mice and adult 4-month-old
mice. The 1-month-old mice exhibited greater maximal
constriction to both light stimuli than 2-month-old mice,
as well as a smaller sustained constriction response toblue light [40], strongly mirroring our present findings inhumans.
The post-illumination pupil response, especially 1.7 s
after light offset, is reported to be entirely driven by melanopsin [13]. Consistent with previous findings in adults[30, 41], post-illumination metrics from our study (i.e.,
AUC and PIPR) were greater in response to blue compared to red light in adolescents. This difference, however, was not observed in our sample of school-aged
children, although the amplitude of the post-illumination
response was at least as large as that of the adolescents.
Coupled with the children’s smaller sustained responseto blue light compared with adolescents, these findings
could suggest continued development of the melanopsindriven pupillary response to evening light during youth.

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

In addition, our findings suggest that the extrinsic input(from rods and cones) to ipRGCs develop differently
from the ipRGCs’ intrinsic, melanopsin-driven, response.
In fact, with development the extrinsic input to ipRGCs
originating from rods and cones appears to decline(lower phasic constriction, slower latency of constriction,and reduced maximum constriction), while the intrinsic
melanopsin-driven input appears to increase or at least
remain stable with development (increased PIPR and
AUC to blue only in adolescents). Whether this reduction in outer retinal input to the PLR would be reflectedin circadian entrainment is unclear and an important
topic for future research. Moreover, data in adults indicate a reduction in the sustained post-illumination pupil
response in the evening compared to daytime [29, 30].
Assuming a similar circadian-dependent pattern in the
response is present in children, examining these outcomes near the peak of the ipRGC-driven response
during the daytime could potentially result in different
outcomes and is another area for future examination.
Although the circadian response to evening lighthas been shown to decrease between pre-pubertal andpost-pubertal children [12], there is a substantial lackof research on typical ipRGC development and changes
in connectivity across childhood. However, researchon outer retinal photoreceptors indicates age-relatedchanges across early and late adulthood. For example, inhuman adolescents and adults, cone density decreases
with axial length and age [42, 43]. Additionally, outerretinal cells undergo extensive reorganization during
normal human aging [44]. The longitudinal assessment ofthese and other mechanisms in early childhood-adolescence is crucial to confirm our findings but also to determine whether our findings of age-related differences in
the PLR reflect structural changes in the retina, connectivity between outer- and inner-retinal photoreceptors,
or maturational changes farther downstream.
Our data were collected utilizing a rigorous protocol designed to control for several confounding variables that could influence the pupillary light reflex. By
remaining indoors and wearing a pair of dark glasses
throughout Day 6 of the protocol, participants were
exposed to significantly less light on the day of theassessment, with less individual and group variability inlight history. Additionally, participants restricted light
exposure between bedtime and wake time throughout
the protocol to provide consistent light/dark timing.
Finally, the pupillary assessment began 1 h before eachparticipant’s habitual bedtime in order to anchor theassessment within a consistent circadian window acrossindividuals. By focusing only on the hour before bedtime, our findings offer a more translatable examination of how the light exposure children and adolescents

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receive before bedtime stimulates the non-visual system, potentially leading to circadian disruption.
Our study has a few limitations. The phase angle
between melatonin onset (a marker of the timing of thecircadian clock) and bedtime grows wider across adolescence [45]. Because we anchored the protocol to habitual
bedtime and did not assess melatonin onset, the exact circadian timing of the pupillary assessment could have differed between individuals and age groups. Furthermore,
adherence to wearing the dark glasses on the final day ofthe protocol was confirmed by self- or parental-reportand could not be verified objectively. Wearing the darkglasses throughout the day and the 1 h dim-light adaptation prior to the PLR measurements could also haveinfluenced the response of the ipRGC, as prior dim-light
adaptation can increase the sensitivity of the circadiansystem to light exposure [46, 47]. Finally, participants inour sample were predominantly reported as Caucasian by
their parents (87.5%). Previous studies with children andadults reported racial differences in the pupillary lightreflex and circadian sensitivity to light [48, 49]. Therefore,
given the lack of diversity in our sample, our findings may
not be representative of the PLR in children and adolescents across different racial and ethnic identities.

Conclusions
In conclusion, in this well-controlled, repeated-measures
study, we highlight differences in the pupillary responseto blue and red light between children (8–9 years) andadolescents (15–16 years). While our findings demonstrate an overall heightened sensitivity to evening light inchildren compared to adolescents, they also suggest continued maturation of the human non-visual photoreception/system throughout development.
Abbreviations
ANOVA	Analysis of variance
AUC​	Area under the curve
ipRGC​	Intrinsically photosensitive retinal ganglion cells
LED	Light emitting diode
OPN	Olivary pretectal nucleus
PIPR	Post-illumination pupillary response
PLR	Pupillary light response
SCN	Suprachiasmatic nucleus
SD	Standard deviation

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s40101-​024-​00363-6.
Supplementary Material 1.
Acknowledgements
We thank the participants and families who took part in this research. We also
thank the students and staff of the Sleep and Development Lab at the University of Colorado Boulder who assisted in data collection.

Hartstein et al. Journal of Physiological Anthropology(2024) 43:16

Authors’ contributions
LEH, MKL, and RPN conceptualized and designed the study. MTD designed
and built the light stimulus apparatuses. LEH collected the data. LEH, MTD,
and RPN analyzed and interpreted the data. LEH and MTD created the figures.
LEH drafted the initial manuscript. All authors revised the drafted manuscript
and read and approved the final manuscript.
Funding
This work was funded by the Eunice Kennedy Shriver National Institute ofChild Health & Human Development (F32-HD103390; R01-HD087707) and the
National Heart, Lung, And Blood Institute of the National Institutes of Health(T32-HL149646). The content is solely the responsibility of the authors anddoes not necessarily represent the official views of the National Institutes of
Health.
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

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Declarations
Ethics approval and consent to participate
All study procedures were approved by the University of Colorado Boulder
Institutional Review Board. Parents provided written, informed consent andchildren/adolescents provided written assent to participate.
Consent for publication
Not applicable.
Competing interests
LEH and MTD have no financial or personal conflicts to declare. MKL reports
receiving travel funds from the Australian Research Council and research support from the National Institutes of Health, beyond the submitted work. RPN
has a patent application for a handheld pupillometer (PCT/SG2018/050204):
Handheld ophthalmic and neurological screening device. The device was not
used in this study.
Author details
1
Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO, USA. 2 Excelitas Technologies, Boulder, CO, USA. 3 Center for Innovation& Precision Eye Health, Department of Ophthalmology, Yong Loo Lin Schoolof Medicine, National University of Singapore, Singapore, Singapore. 4 ASPIRE
Research Program, Singapore Eye Research Institute, Singapore, Singapore.
5
Duke-NUS School of Medicine, Singapore, Singapore. 6 Department of Biomedical Engineering, College of Design and Engineering, National Universityof Singapore, Singapore, Singapore.

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22.

Received: 9 February 2024 Accepted: 4 June 2024

23.

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