University of Groningen Grasping light Lok, Renske

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Grasping light

Lok, Renske

DOI:

10.33612/diss.173352710

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Publication date:

2021

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Citation for published version (APA):

Lok, R. (2021). Grasping light: Mental and physiological responses to illumination. University of Groningen.

https://doi.org/10.33612/diss.173352710

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CHAPTER 3

WHITE LIGHT DURING

DAYTIME DOES NOT

IMPROVE ALERTNESS IN

WELL-RESTED INDIVIDUALS.

Renske Lok

1,2

_{, Tom Woelders}

1

_{, Marijke C.M. Gordijn}

1,3

_,

Roelof A. Hut

1

_{, Domien G.M. Beersma}

1

1_{University of Groningen, Chronobiology unit, Groningen Institute for} Evolutionary Life Sciences, PO box 11103, 9700CC, Groningen, the Netherlands.

2_{University of Groningen, Campus Fryslân, Wirdumerdijk 34, 8911 CE,} Leeuwarden, the Netherlands.

3_{Chrono@Work, B.V., Friesestraatweg 213, 9743 AD, Groningen, the} Netherlands.

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Abstract.

Broad-spectrum light applied during the night has been shown to affect alertness in a dose-dependent manner. The goal of this experiment was to investigate whether a similar relationship could be established for light exposure during daytime. 50 healthy participants were subjected to a paradigm (07:30 AM - 5:30 PM) in which they were intermittently exposed to 1.5 hours of dim light (DL,

<

10 lux) and 1 hour of experimental light (EL, ranging from 24 to 2000 lux). The same intensity of EL was used throughout the day, resulting in groups of 10 subjects per intensity. Alertness was assessed with subjective and multiple objective measures. There was a significant effect of time of day in all parameters of alertness (p

<

0.05). Significant dose-response relationships between light intensity and alertness during the day could be determined in a few of the used parameters of alertness on some times of the day; however none survived correction for multiple testing. We conclude that artificial light applied during daytime at intensities up to 2000 lux does not elicit significant improvements in alertness in non-sleep deprived subjects.

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3 Introduction.

Light is known to elicit both image forming and non-image forming (NIF) responses. One of these NIF-responses is improved alertness, which so far has been established especially during the night 4,78,81–85,88_{. Humans are diurnal,} with optimal psychological and physiological performance during daytime 98–100_. Alertness is known to affect many functions, such as performance, psychological and physiological well-being, caloric intake and pain sensitivity 95,96,101,102_{. Thus,} displaying optimal alertness is beneficial in many facets of everyday life 95,98–100,102_. It is essential to measure such NIF-responses during the day, as it is unclear whether daytime effects of light on alertness are similar to nighttime effects (a full discussion of daytime effects of light on alertness can be found in this issue, see 98–100,170,180,197_{and positive effects of daytime light exposure on other NIF-responses} (such as sleep, health, mood and mental disorders) have been reported 198–201_{. The} aim of this study was to determine dose-response relationships between several measures of alertness and broad-spectrum light exposure at different times of day. To increase accuracy, alertness was assessed with a multi-measure approach, in which subjective sleepiness (measured with the Karolinska Sleepiness Scale; 139_{), performance on the Go-NoGo task}202_{and various physiological correlates of} alertness (such as skin temperature 69_{and blink measurements}147_{) were taken} into account 28,81,137,157,159,160,162_.

Materials and methods.

Subjects. Participants were 50 healthy, non-sleep deprived subjects (25 female, 25

male) between the ages of 20 and 30 years (average ± SEM; 23.02 yrs ± 0.29). All participants gave written informed consent and received financial compensation for participation. The study protocol, screening questionnaires and consent forms were approved by the medical ethics committee of the University Medical Center Groningen (NL54128.042) and were in agreement with the Declaration of Helsinki (2013).

Participant’s health was assessed via an in-house developed general health questionnaire. As an indication of sleep timing, chronotype was assessed via the Münich Chronotype Questionnaire (MCTQ; Roenneberg et al., 2003). To determine baseline sleep quality, participants completed the Pittsburgh Sleep Quality Index (PSQI; Buysse et al., 1989). Participants reported no health problems, were intermediate chronotypes (MSF_scbetween 3.88 - 6.17, average ± SEM; 4.80 ± 0.08), and did not report more than mild sleep problems (PSQI

<

12, average ± SEM; 4.06 ± 0.31). Exclusion criteria were: 1) chronic medical conditions or the need for (sleep)medication use, 2) shift work three months before participation, 3) having travelled over multiple time-zones within two months before participation, 4) smoking, 5) moderate to high levels of caffeine intake (more than 4 cups per

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day), 6) excessive use of alcohol (

>

3 consumptions per day), 7) use of recreational drugs in the last year, 8) a body mass index outside the range of 18 to 27, a body weight of less than 36 kg, 9) inability to complete the Ishihara color blindness test

205_{without errors upon arrival. The estimated average ± SEM of caffeine intake per}

day was 0.85 ± 0.12 cups per day for the included participants.

Protocol. Subjects arrived at the human isolation facility of the University of

Groningen the evening before the experimental day and remained in their individual rooms in dim light (DL). Computers were covered with blue light blocking foil and set to minimal intensity. Both the spectral composition and illuminance of experimental light (EL) was measured at the start of the experiment for every individual and included the amount of light coming from the computer screen. A practice test session was performed in which participants were monitored, to verify sufficient understanding of the tasks. After completion, individuals were equipped with DS1922L Ibuttons® (Thermochron, Australia) for measuring skin temperature on the right and left clavicles, middle fingers and ankles. Participants were instructed to go to bed at 11:30 PM and were woken up the next morning at 7:30 AM under dim light conditions (i.e.

<

10 lux). During the rest of the day, participants were exposed to four consecutive cycles of 1.5h of DL followed by 1h of experimental light (10h in total; see Fig. 1). Participants were subjected to 1 hour of EL, since according to literature, effects of light on alertness can already be present after only 30 minutes of light exposure 4,171_{. DL exposure of 1.5 hour was}

chosen to allow for a return to baseline alertness after each hour of EL exposure. During every cycle, two test sessions were performed in DL (18 and 78 min after onset of DL) and two test sessions were performed in EL (18 and 48 min after onset EL).

Figure 1. Schematic representation of the experimental design. DL exposure lasted for 90 minutes, EL

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3

A test session comprised of participants completing the Karolinska Sleepiness

Scale (KSS; Åkerstedt & Gillberg, 1990), followed by a 6-minute eye blink recording. During this task, subjects had to remain seated as still as possible and focus on a fixation mark, while wearing glasses fitted with an infrared emitting diode and photosensitive diode. Differences in infrared reflectance between the eyelid and eyeball were used to determine several blink parameters 147_{. Thereafter,} participants completed a 5-min auditory Go-NoGo task (performed in Visual Studio 2015), to assesses executive control 202_{. Skin temperature was measured} throughout the experiment at a sampling frequency of 60 seconds. Iso-caloric snacks were provided 30 minutes after each DL onset. Basic metabolic rate (BMR) estimation was used to calculate caloric value of the snack as follows: BMR = ((10 * weight(kg)) + (6.25 * length(cm)) - (5 * age(yrs)) + 5)) for males and BMR = ((10 * weight(kg)) + (6.25 * length(kg)) - (5 * age(yrs))-161)) for females 206_. Two thirds of this recommended daily caloric intake was divided over 4 snacks and provided during the 10h experiment. Snacks were served with caffeine-free tea or water.

Light Exposure. Polychromatic white DL (

<

10 lux) was provided via ceiling-mounted Philips fluorescent tube lights (see Fig. S1 and Table S1 for spectral composition and illuminance values). EL was delivered by a portable polychromatic white light source, consisting of a modified Philips Energy Up light (HF3419/02, Philips, Drachten, tQhe Netherlands) in which two white light emitting diodes (LEDs) were replaceQQd with blue LEDs. This was necessary to ensure sufficient stimulation of all photoreceptor classes. The EL lamp was placed on the desk in front of the participants at a distance of 20 cm. Intensities were chosen to span the full range of the dose-response curve of alertness-enhancing effects of light, based on night- and daytime data 196_{. Intensities were therefore set to 24, 74, 222, 666 or} 2000 photopic lux corresponding to 21, 67, 219, 642, 1933 melanopic lux 8_(Table S1). Each participant was exposed to one single intensity setting throughout the 4 EL blocks on the experimental day. As each subject participated for only 1 day, the experiment followed a within (time of day) and between (intensity) subjects design (10 subjects per intensity). The intensities of EL were evenly distributed over gender and day of the week (Monday-Friday). The experiment was conducted between May and September 2016, local time is expressed as GMT+2 (thus using daylight savings time).

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Data pre-processing: blink parameters. Signals from the photosensitive diode

of the blink measuring glasses were stored on a computer at a sampling frequency of 200 Hz. Analyses were based on previously described methods 147_{. Blink} parameters were determined using the native MATLAB R2015b Signal Processing Toolbox function ‘findpeaks’. Blink frequency (defined as the number of peaks per minute), blink duration, closing time, reopening time and total time that the eyelid was fully closed were assessed. Blinks were characterized by differences in infrared reflectance between the opened and closed eyelid, recognized by a u-shaped infrared reflectance peak over time. Blink duration was determined as follows: a baseline reflectance value was calculated for each blink, existing of the average reflectance of the completely opened eye in a time window of 500 ms, before the signal amplitude reached 10% of the amplitude of the reflectance peak. Linear regression lines were fitted to both sides of the inverted u-shaped blink form. Blink duration (ms) was then determined as the time difference between the baseline-crossings of both regression lines. The timing of the intersection of both regression lines was considered as the timing of peak reflectance (i.e. full closure of the eyelid). Eyelid closing and reopening time was then determined by calculating the time-interval between the timing of peak reflectance and the closing and reopening (i.e. 10% deviations from baseline) of the eyelids. The total time that eyelid was fully closed was determined as the time during which the signal remained higher than 90% of peak amplitude 147_{. Eyelid closures not} fulfilling the following criteria were excluded: 1) a blink duration of 50–500 ms; 2) a closing time of

<

150 ms; 3) a reopening time

<

150 ms 147_{, to exclude non-blink} closures 207,208_{. The first and last 30 seconds of each 6-minute measurement were} omitted from analysis, to exclude possible noise from filling in the questionnaire or anticipation of the end of task. Decreased blink frequency, blink duration, eyelid closure time, the total time that the eyelid was closed and eyelid reopening time have been shown to be related to an increase in alertness 147_.

Data pre-processing: Go-NoGo. Tests were performed on a HP Compaq 8200

Elite Convertible Minitower PC with KB-0316 keyboard. For every individual, errors of omission were defined as response latencies greater than the average of all test sessions + two standard deviations, anticipation errors as response latencies shorter than the average of all test sessions - two standard deviations and commission errors as responding to non-target stimuli. Other parameters of interest were median RT, average RT, the average of the 10% fastest and 10% slowest RTs, and the average RT in the first and last minute of the 5-minutes test. A decrease in RT, and/or omissions, commissions or anticipation indicate an increase in alertness.

Data pre-processing: Skin temperature. Skin temperature data were collected

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3

sampling interval, 0.0625 ºC resolution, 0.5 ºC accuracy). Outliers with absolute consecutive temperature change exceeding 2ºC were omitted. Distal skin temperature was calculated as the average temperature of fingers and ankles. Proximal skin temperature was determined as the average temperature of both clavicles. The distal-proximal gradient (DPG) was calculated as the distal minus proximal skin temperature 209_{. For construction of dose-response curves, the}

distal- and proximal skin temperature and DPG were calculated as the average of 18 data points (i.e. 18 minutes) immediately prior to the start of each test session. Higher proximal- and lower distal skin temperature and larger DPG are associated with higher alertness.

Statistics. Mixed linear models were constructed in RStudio (version 1.0.136)

for each alertness parameter, with that parameter as the dependent variable. Independent variables were time of day and light intensity. Fixed effects consisted of time of day (as categorical variable), intensity condition and the interaction term. To control for between-subject variation, subject ID was included as a random effect. Sigmoidal curves were fitted to the data obtained in each test session separately, to determine dose-response relationships.

To this end, an adapted version of the Naka-Rushton equation

210,211

_was

used, with Y

,

in which Pmin represents the minimum – and Pmax the maximum of the

chosen parameter, a represents the half-maximal response constant

(I50) and b the slope parameter. Significance was determined with the

Microsoft Excel 2010 Solver function, using the Generalized Reduced

Gradient Nonlinear function for smooth nonlinear functions, which uses

the gradient of slope of the objective function as the input values and

determines that it has reached an optimum solution when the partial

derivative equals zero

. A definition of a critical two-sided alpha value of

0.05 was maintained for all statistical tests.

Results.

Linear model analysis revealed that there were no significant differences in age (F1,48=2.37, p=0.13) MSFsc (F1,48=0.07, p=0.79), PSQI score (FF1,48=0.11, p=0.74) and

caffeine intake (F_1,48=0.53, p=0.47) between the subjects in the different EL intensity groups.

The time courses of subjective sleepiness score, median reaction time, blink duration and DPG, all indicate a pattern over time of day independent of DL or EL light exposure (Fig. 2). Time of day was found to be significant in subjective sleepiness score (

χ

²=111.7, p

<

10-15_{, n=50, df=15), reaction time (}

χ

2_{=60.5, p}

<

₁₀-6_,

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p

<

10-15_{, n=50, df=15). A signifi cant interaction between time of day and light}

intensity was found in subjective sleepiness score (

χ

2_{=90.5, p}

<

_{0.007, n=50, df=60)}

and DPG (

χ

2_{=172.8, p}

<

₁₀-13_{, n=50, df=15).}

Signifi cant eff ects of time of day were also established for other direct and indirect measurements of alertness. The time course of the 10% fastest- and slowest reaction times, average overall reaction time, average reaction time in the fi rst and -last minute of the task, blink frequency, eyelid closing- and reopening time, total time that the eyelid is closed and the proximal- and distal skin temperature are shown in Fig. S2-S4. In the Go-NoGo test, too few errors of anticipation, omission and commission were made to allow for further statistical analyses.

Figure 2. Time course of parameters of alertness. Depicted are subjective alertness (A), median reaction time (B), blink duration (C) and distal-proximal gradient (D). N=10 per intensity group. Data represent mean ± SEM. Timing of DL and EL is indicated by the grey and white shaded areas respectively.

To reveal short term light-exposure eff ects independent of long term trends, time of day patterns were eliminated as follows. For each individual, a linear interpolation was calculated between the last data point in DL before EL was turned on of one block and the last point in DL before EL was turned on in the next block (Fig. S5). This linear interpolation was interpreted to follow the long term change in alertness over time, including circadian regulation of alertness. The vertical distance of every data point relative to the linear interpolation was then determined. This procedure served as a correction for time of day for all

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3

parameters of alertness. For the last test session, the slope of the interpolation from the previous session was assumed, as no DL data points were available after termination

of the last EL episode. Data were normalized by z-transformation at the level of

participant ID to eliminate between-subject variation. The various correlates of alertness were plotted against EL intensities (photopic lux). When tested for dose response relationships between EL intensity and alertness parameters, light intensity was not found to signifi cantly contribute to subjective sleepiness score, reaction time, blink duration or DPG (Fig. 3, table S2). At 15:18, a signifi cant dose-response relationship was found between photopic lux and blink frequency (F4,46=3.06, p

<

0.04; Fig. 4, table S3), with fewer blinks occurring with increasing light

intensity. When correcting for multiple testing using the Bonferroni correction, none of the fi tted curves constructed for 10% fastest and 10% slowest reaction times, overall average reaction time, average reaction time in the fi rst- and last minute, blink frequency, eyelid closing- and reopening time, total time that the eyelid was closed, proximal- and distal skin temperature a signifi cant contribution of light intensity was found, at any time of day (Fig. S6-S8, table S3).

Figure 3. Relationships between illuminance (lux) and parameters of alertness. Presented are subjective alertness (A), median reaction time (B), blink duration (C) and distal proximal gradient (D). All values were z-transformed and corrected for time of day eff ects. Data represent mean ± SEM. Circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off . Figures for other correlates of alertness can be found in S5-S7.

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Figure 4. Plots of signifi cant dose-response relationships between illuminance (lux) and parameters of alertness. Depicted is blink frequency at indicated time of day. All values were z-transformed, corrected for time of day eff ects and averaged over time of day. Open circles indicate individual data points and dashed lines represent the calculated fi t of the dose-response curve.

As values were corrected for time of day, one composite score per individual could be calculated (irrespective of time of day), SA signifi cant dose-response relationship was found between light intensity and sleepiness (F4,45=3.10, p=0.036)

18 minutes after the light had been turned off , with decreased sleepiness when intensity increases. Saturation seems to appear at a light intensity of 75 lux (Fig. 5 and 6). Median reaction time, blink duration and DPG did not show signifi cant dose-dependent changes (Fig. 5, table S4). A signifi cant dose-response relationship was observed after 18 minutes of light exposure in eyelid closure time (Fig. 6, F4,45=3.25, p=0.030), refl ecting slower closure times as light intensity increases.

When correcting for multiple testing with the Bonferroni correction, none of the fi ts passed the signifi cance threshold (

α

corrected=0.02). None of the fi ts of other

parameters of alertness (10% fastest and 10% slowest reaction times, overall average reaction time, average reaction time in the fi rst minute, blink frequency, eyelid reopening time, total time that the eyelid was closed, proximal- and distal skin temperature) reached signifi cance (Fig. S9-S11, table S4).

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3

Figure 5. Relationships between illuminance (lux) and parameters of alertness. Presented are subjective alertness (A), median reaction time (B), blink duration (C) and distal-proximal gradient (D). All values were z-transformed, corrected for time of day eff ects and averaged over time of day. Data represent mean ± SEM, parameter values and signifi cance levels of dose response curve fi ts are in table S4. Circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off . Figures for other correlates of alertness can be found in supplementary information (Fig. S8-S10).

Figure 6. Plots of dose-response relationships between illuminance (lux) and parameters of alertness. Shown are averaged eyelid closing time (A) and sleepiness (B) after diff erent times of light exposure. All values were z-transformed, corrected for time of day eff ects and averaged over time of day. Dose-response curve parameter values and signifi cance levels can be found in table S4. Open circles indicate individual data points and dashed lines represent the calculated fi t of the dose-response curve.

Discussion.

The goal of this experiment was to determine dose-response curves for several measures of alertness in response to polychromatic white light during daytime. Our results show that, although a few correlates of alertness show signifi cant dose response relationships at certain times of day, no such relationships were observed at other times of day. Moreover, multiple other objective and subjective correlates did not show dose-dependent changes in response to light during any time of day. In fact, eff ects of light on alertness were found to be small, if present

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at all. Therefore, the dose-response relationship between light and subjective and objective correlates of alertness at night 78_{could not be confirmed during daytime.} With the many tests performed, it is possible that the few significant correlations occurred by chance. Correcting for multiple testing requires independent testing and it is plausible that multiple correlates of alertness are not independent. Therefore, such a correction (for example the Bonferroni correction) is too conservative, leading to a high rate of false negatives. Hence, if it is assumed that all significant dose-response relationships indeed depict the true dose-response relationship, then it should be noted that subjective alertness displays a relationship in the same direction as established during the night 78_{, in which improved alertness} occurred with increasing light intensities. Noteworthy is the fact that saturation of this response during daytime seems to appear at a light intensity of 75 lux, while saturation at night starts at a light intensity of approximately 110 lux 78_{. The circadian} system is thought to be more sensitive to light at night, therefore saturation of daytime subjective alertness at a lower light intensity might imply that the phase response relationship between light and alertness may differ from that known for light and phase shifts of the circadian pacemaker 212_{. Eyelid closing time showed} decreased alertness or increased sleepiness at higher light intensities. This lack of consistency, combined with the fact that the majority of parameters at most times of the day do not show a significant dose dependent relationship with light intensity, suggests that effects of light on alertness during daytime are very small, if present at all. This interpretation of the data is supported by a different study, in which 60 subjects were exposed to light at similar intensities as were employed here (20 to 2000 lux; 90_{). Although employing a different experimental design and} using different measures of alertness, their results also indicate that effects of broad-spectrum white light on alertness are not present during daytime 90_{. Most} importantly, the authors conclude that there is no dose-response relationship between broad-spectrum light intensity and alertness during daytime 90_.

The two process model of sleep-wake regulation indicates that sleep pressure is high during the evening hours, since homeostatic sleep pressure increases only with elapsed time awake 47_{. However, ‘sleepiness’ receives additional circadian} influence when the circadian system promotes wakefulness towards the end of the waking period 47,213_{. As the drive for wakefulness and corresponding alertness} levels, influenced by both processes, is relatively unresponsive to light-exposure during daytime as our results suggest, it is possible that a ceiling effect of alertness is present at that time. Indeed, light has been shown to increase alertness during the day in mildly sleep-deprived or mentally fatigued individuals 188,189_{. On the other} side of the spectrum, chronic sleep deprivation may compromise the system too severely beyond recovery. Therefore, an inverted u-shaped relationship between alerting effects of light and the level of fatigue may be expected.

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3

There are, however, studies indicating improvements in alertness in response

to light during daytime in well-rested individuals 86,87,193,214_{. Methodological} differences among these reports complicate comparison between studies with positive, neutral, mixed or negative results 197_{. In these studies, large variations in} terms of spectra and intensities exist between light devices employed. Differences in spectral composition of broad band white light might cause large differences in alertness due to opposing contributions of different cone types to NIF responses in the human retina 167,215_{. In addition, duration of light exposure and} time of day varies between studies, as well as subject inclusion criteria, sample sizes and experimental protocols, in which some implemented sleep restriction or deprivation and others did not. Control conditions, often comprising of ‘dim

light’, vary as well, with studies reporting conditions of

<

1 lux and others using 200 lux 87,216_{. Other factors complicating comparison are photoperiodic effects} on NIF-responses, such that time of year may play a role. Indeed, greater effects of artificial light have been reported under short photoperiods in both hamsters 217_{and humans}218_{. Effects of previous light history on light sensitivity might also} affect NIF-responses, with reports showing more melatonin suppressionafter lower levels of previous light exposure 219_{and adaptation to 2 weeks of} blue-filtered light exposure reflected in normalizing of the response of melatonin suppression to a light pulse 220_{. Even if time of year might be the same, differences} in entrainment may also compromise light triggered responses. In this study, we did not collect Actigraphy data preceding the in-lab part of the experiment, and we therefore cannot be absolutely sure that all subjects were entrained in the same way. However, we did select on chronotype (MSFsc), which has been shown to correlate well with the clock phase marker dim light melatonin onset 221_. Other individual differences might complicate matters even further. In responses to monochromatic light, Vandewalle and colleagues (2006) found significant improvements in subjective alertness during daytime, which was correlated to posterior thalamic responses only in individuals who showed an alerting response to light. Since inclusion criteria were the same for all participants, this indicates that there are individual differences in light sensitivity, such that some individuals respond to light whereas others do not 109_{. Interindividual differences in genetic} makeup may account for these differences in light sensitivity, which might partly be explained by polymorphisms in the PER3 clock gene 222_{. Another factor that may} explain these interindividual differences, is the interindividual variation in locus coeruleus (LC) activity, which could affect both baseline alertness and alerting effects of light 223_{. Individual differences in melanopsin signaling have also been} reported to affect NIF-responses 224_{. Together, these studies suggest that there is} individual variation in light sensitivity which might influence NIF-responses such as alerting effects of light. During daytime, when the circadian system is relatively insensitive to light, individual differences in light sensitivity might contribute significantly to (a lack of) light response as compared to nighttime, when the circadian system promotes sleep.

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Rhythmicity of the circadian system could underlie differences in alerting effects of light between night and day. There are several physiological factors that are under circadian control, of which one is pineal melatonin secretion. Melatonin secretion peaks during the night and is virtually absent during the day 35_{. Nighttime exposure} to both polychromatic white- and monochromatic blue light have been shown to improve alertness while at the same time suppressing melatonin 86,116,163,171,225,226_. Correlations between melatonin suppression, subjective and objective measures of alertness have been demonstrated 81,107_{. However, some studies that did} not find effects of melatonin suppression on subjective sleepiness 227_{and other} studies induced alerting effects without suppressing melatonin 95,177_{or found} alerting effects of light at times of day when melatonin is absent 86,188_{. It has been} shown that daytime administration of (super pharmacological levels) exogenous melatonin may induce sleepiness 228_{. A partial causal role of melatonin suppression} in mediating the alerting effects of light 225,229_{might explain our findings that} daytime light exposure does not result in increased alertness because there is no melatonin production during daytime. Alternatively, the sensitivity to alerting effects of light need not necessarily be directly related to melatonin suppression, but may also be under direct circadian control such that light is less effective in inducing alertness during clock phases corresponding to the active period. In summary, there are several factors that could affect results of experiments investigating NIF-effects of light, which are usually not taken into account. These factors may either have contributed to previously reported positive results, or to the current lack of effect. This may indicate that alerting effects of light during daytime can only occur under highly specific or controlled circumstances and possibly only in certain individuals, which would limit practical applications. The number of experiments reporting positive effects of polychromatic white light on alertness during daytime are limited, as are the studies reporting negative results 197_{. The possibility that some of these results might be chance observations should} not be neglected, given all factors provided above. A publication bias towards experiments reporting positive effects of light and alertness may exist, since negative results are usually less often published.

Our results suggest a pattern in both subjective and objective parameters of alertness over the course of the day, with relatively low levels of subjective alertness upon awakening, relatively high levels in the (early) afternoon and a decrease in alertness towards the end of the day. This was unaffected by the light interventions. A similar pattern has been shown in several studies 230,231_{. In} a recent field study with a large sample size (n = 431), Åkerstedt and colleagues showed similar patterns of subjective alertness over the course of the day as we report here 115_{, although no light-based interventions were applied in that study.} These findings support the notion that patterns of subjective alertness over the course of the day are independent of light manipulation.

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3

In conclusion, our results indicate that 1-hour polychromatic white light

pulses administered at different times of day do not improve alertness in a dose-dependent manner in well-rested individuals. Whatever the underlying mechanism, we conclude that the alerting effect of light during the day is much smaller than during the night, if present at all.

Acknowledgements.

The authors would like to thank student Dick Ameln for his efforts in designing and constructing the glasses that were used to measure blink-parameters and student Nina Ranchor for her assistance in collection of the data.

Funding.

This research was funded by the University of Groningen Campus Fryslân (grant #01110939; cofinanced by Philips Drachten and provincie Fryslân).

Conflict of interest.

The authors have declared the following potential conflict of interest with respect to the research, authorship, and/or publication of this article: Philips Drachten has made an in-kind contribution to the experiment. Dr. Gordijn reports receiving consultancy fees from Philips Consumer Lifestyle, not related to the submitted work.

Data availability.

Original data, analyses, and R-codes can be accessed by contacting the corresponding author. The complete data-set (original and analyzed) and R-codes are also available at the data repository at the University of Groningen.

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Supplementary information

Table S1: Photometric properties of background- and experimental light according to the Lucas file8

Type Color temperature (K) Illuminance (lux) Cyanopic (lux) Melanopic (lux) Rhodopic (lux) Chloropic (lux) Eyrthropic (lux) Background 2500 10 3 4 5 8 10 Experimental light 5800 24 20 21 23 27 29 Experimental light 5800 74 66 67 68 73 74 Experimental light 5800 222 206 219 221 230 226 Experimental light 5800 666 584 642 644 661 647 Experimental light 5800 2000 1744 1933 1934 1975 1925

Figure S1: Spectral composition of background light (dashed line) and experimental light (solid line). Illuminance was measured on the vertical plane at the level of the eye. Background light was generated by ceiling-mounted fluorescent light TL tubes. Experimental light consisted of a modified Philips Energy Up light, in which two white LEDs had been substituted by two blue LEDs.

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3

Figure S2: Time course of performance correlates of alertness, measured with the Go-NoGo task. Depicted from top to bottom are the 10% fastest (A)- and slowest (B) reaction times, average overall reaction time (C), average reaction time in the first (D)- and last minute (E). N=10 per intensity group. Data represent mean ± SEM. Timing of DL and EL is indicated by the grey and white shaded areas respectively.

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Figure S3: Time course of physiological correlates of alertness, measured with infrared radiation detection glasses. Depicted are the blink frequency (A), eyelid closing time (B), total time that the eyelid is closed (C) and the reopening time (D). N=10 per intensity group. Data represent mean ± SEM. Timing of DL and EL is indicated by the grey and white shaded areas respectively.

Figure S4: Time course of skin temperature, measured with Ibuttons. Depicted are the proximal (A)- and distal (B) skin temperature. N=10 per intensity group. Data represent mean (black line) ± SEM(grey). Timing of DL and EL is indicated by the grey and white shaded areas respectively.

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3

Figure S5: Hypothetical data illustrating the applied correction for time of day effects. A linear interpolation was used to determine the slope between the last point in DL of one block and the last point in DL in the next block (indicated by dashed line). The vertical distance of every data point relative to this interpolation was determined, at the level of participant ID.

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Figure S6: Relationship between illuminance and reaction time on the Go-NoGo task. Depicted are the 10% fastest (A)- and slowest (B) reaction times, average reaction time (C), average reaction time in the fi rst (D) and -last minute (E) against intensity (lux). Intensity is depicted on a logarithmic scale. All reaction time parameters are z-transformed and corrected for time of day eff ects at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off .

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3

Figure S7: Relationship between illuminance and eye-parameters. Depicted are blink frequency (A), eyelid closing time (B), total time that the eyelid was closed (C) and eyelid reopening time (D) against intensity (lux). Intensity is depicted on a logarithmic scale. Eye parameter values are z-transformed and corrected for time of day at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off.

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Figure S8: Relationship between illuminance (depicted on a logarithmic scale) and distal (A)- and proximal (B) skin temperature. Values are z-transformed and corrected for time of day eff ects at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off .

Figure S9: Relationship between illuminance (depicted on a logarithmic scale) and fastest (A)- and slowest (B) reaction times, average reaction time (C) and fi rst (D)- and last minute (E) reaction time. Values are averaged over all times of day, z-transformed and corrected for time of day eff ects at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off .

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3

Figure S10: Relationship between illuminance (depicted on a logarithmic scale) and blink frequency (A), eyelid closing time (B), the total time that the eyelid is closed (C) and reopening time (D). Values are averaged over all times of day, z-transformed and corrected for time of day eff ects at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off .

Figure S11: Relationship between illuminance (depicted on a logarithmic scale) and distal (A)- and proximal (B) skin temperature and the distal-proximal gradient. Values are averaged over all times of day, z-transformed and corrected for time of day eff ects at the level of participant ID. Data represent mean ± SEM. Black circles with solid lines represent data collected after 18 minutes of EL exposure, squares with dashed lines follow data collected after 48 minutes EL exposure and triangles with dotted lines represent data collected 18 minutes after EL was turned off .

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Table S2

: Dose response curves were fitted with a modified version of the N

aka-Rushton equation:

.

Values of the best fit are shown for subjective alertness, media

n reaction time and blink duration at different times of day, du

ring- or after EL exposure.

Pmin Pmax a b p F SS Df Sleepiness score 09:00 – 10:00 EL 18 min. EL exposure -0.20 2.38 1.31 27.03 0.72 0.45 65.95 46 48 min. EL exposure -2.28 0.45 4.04 6.17 0.60 0.62 62.71 46

18 min. after EL was turned off

-2.52 0.14 4.07 4.50 0.34 1.14 47.60 46 Sleepiness score 11:30 – 12:30 EL 18 min. EL exposure 0.041 2.96 1.27 48.18 1.00 0.01 46.28 46 48 min. EL exposure -0.15 0.67 2.61 0.27 1.00 0.01 59.31 46

-1.34 1.66 1.60 0.44 0.87 0.24 34.06 46 Sleepiness score 14:00 – 15:00 EL 18 min. EL exposure 0.21 0.52 2.30 45.71 0.74 0.41 37.17 46 48 min. EL exposure 0.001 3.38 0.65 1.43 0.70 0.47 63.47 46

0.17 2.25 1.36 31.85 0.13 1.96 37.07 46 Sleepiness score 16:30 – 17:30 EL 18 min. EL exposure -0.54 0.40 3.01 46.07 0.25 1.41 73.46 46 48 min. EL exposure -1.77 3.16 0.87 0.25 0.98 0.06 116.18 46

Median reaction time 09:00 – 10:00 EL

18 min. EL exposure -1.06 2.14 1.31 -0.25 0.96 0.11 38.83 46 48 min. EL exposure -1.07 0.51 1.30 -25.05 0.78 0.35 27.22 46

-1.19 1.22 1.11 -0.56 0.78 0.36 21.67 46

18 min. EL exposure -1.84 0.88 1.20 -0.67 0.513 0.78 18.23 46 48 min. EL exposure -0.53 -0.12 -64.88 1.87 0.26 1.37 15.44 46

-0.65 -0.15 1.86 -69.71 0.19 1.65 18.69 46

18 min. EL exposure -2.08 -0.27 1.24 -8.06 0.32 1.18 29.36 46 48 min. EL exposure -1.45 -0.29 1.31 -9.49 0.35 1.11 21.43 46

-1.73 -0.31 1.27 -14.17 0.54 0.75 20.08 46

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3

Pmin Pmax a b p F SS Df

18 min. EL exposure -0.42 -0.36 0.79 -12.04 1 2.71 e-5 55.58 46 48 min. EL exposure -3.34 -0.35 0.73 -13.27 1 2.56 e-6 80.52 46 Blink duration 09:00 – 10:00 EL 18 min. EL exposure -0.20 0.72 1.26 -0.10 1.00 5.71 e-4 69.20 46 48 min. EL exposure -1.79 2.24 2.68 0.61 0.75 0.40 65.93 46

-1.91 1.84 2.14 0.50 0.79 0.35 47.21 46 Blink duration 11:30 – 12:30 EL 18 min. EL exposure -1.49 1.92 2.48 0.15 1.00 0.02 71.73 46 48 min. EL exposure -1.68 2.41 2.33 -0.29 0.96 0.09 68.26 46

-1.93 1.83 1.99 0.24 0.97 0.09 41.89 46 Blink duration 14:00 – 15:00 EL 18 min. EL exposure -0.97 0.30 1.20 -3.84 0.65 0.55 36.47 46 48 min. EL exposure -1.98 1.84 3.17 0.39 0.92 0.16 63.45 46

-2.37 2.62 1.42 0.47 0.71 0.46 54.17 46 Blink duration 16:30 – 17:30 EL 18 min. EL exposure -2.27 0.11 1.24 -13.08 0.79 0.35 81.68 46 48 min. EL exposure -3.96 3.70 e10-3 1.33 -21.04 0.37 1.07 171.44 46

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Table S3 : Dose response curves were fitted with a modified version of the Naka-Rushton equation: . Values of the best fit are shown for all other parameters of alertness, during- or after EL exposure. Significant values are indicated with *, but only last minute

reaction time reached significance at 12:48, 18 minutes after EL

had been turned off, after correcting for multiple comparisons(

α

corrected =4.55e -3). Pmin Pmax a B p F SS Df

10% fastest reaction time 09:00 – 10:00 EL

-1.43 -0.15 0.41 -16.90 0.87 0.23 14.19 46

-1.81 -0.20 0.41 -16.90 0.99 3.61 e-3 21.88 46

-1.50 0.18 0.96 -2.00 0.48 0.83 18.6 46

10% slowest reaction time 09:00 – 10:00 EL

18 min. EL exposure -0.16 1.55 3.15 -0.60 0.92 0.14 29.28 46 48 min. EL exposure 0.35 1.65 3.48 -22.22 0.68 0.50 21.60 46

0.14 1.32 3.33 -66.31 0.38 1.03 18.44 46

-1.26 -0.14 1.30 -4.40 0.29 1.25 18.53 46

-1.65 -0.31 1.160 -6.68 0.53 0.73 15.47 46

18 min. EL exposure -0.77 0.23 9.05 -0.26 0.99 0.01 49.45 46 48 min. EL exposure -0.81 0.11 1.77 0.08 0.99 6.15 e-3 74.77 46

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3

Pmin Pmax a B p F SS Df

Average reaction time 09:00 – 10:00 EL

18 min. EL exposure -0.02 1.22 3.47 -0.19 0.99 7.91 e-3 33.54 46 48 min. EL exposure -0.02 0.88 4.09 -0.38 0.99 0.02 24.41 46

0.142 1.39 3.42 -53.47 0.92 0.15 19.62 46

18 min. EL exposure 0.83 -1.42 2.02 0.47 0.79 0.34 15.11 46 48 min. EL exposure -1.52 -0.20 1.11 -4.86 0.39 1.01 12.54 46

-1.23 -0.20 1.33 -5.54 0.24 1.42 16.29 46

-0.23 -0.23 -3.53 -1.56 0.42 0.94 15.87 46

18 min. EL exposure -0.77 0.03 1.68 -0.46 0.99 0.01 50.85 46 48 min. EL exposure -2.26 1.60 1.31 -2.73 e-3 1.00 6.58 e-6 76.61 46

First minute reaction time 09:00 – 10:00 EL

-1.28 0.19 1.30 -35.54 0.86 0.23 17.10 46

18 min. EL exposure -0.83 0.71 1.44 0.23 0.98 0.04 16.53 46 48 min. EL exposure -0.53 0.24 1.25 0.11 0.99 2.40 e-3 13.64 46

-0.85 0.30 0.71 -0.18 0.99 0.01 16.48 46

-1.62 0.19 1.34 -23.99 0.21 1.52 21.14 46

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Last minute reaction time 09:00 – 10:00 EL

18 min. EL exposure 0.09 0.48 2.80 66.75 0.59 0.63 29.76 46 48 min. EL exposure 0.17 0.76 0.75 32.37 1.00 3.75 e-3 23.07 46

-0.78 0.85 0.61 0.19 1.00 0.05 21.57 46

18 min. EL exposure -1.59 0.48 1.04 -0.60 0.83 0.29 21.81 46 48 min. EL exposure -1.35 -0.19 1.30 -14.75 0.32 1.19 13.45 46

-0.579 -0.08 1.83 -32.94 0.12 2.00 13.77 46

18 min. EL exposure -1.35 0.01 1.22 -6.73 0.57 0.66 30.11 46 48 min. EL exposure -0.92 -0.08 1.06 -6.86 0.97 0.07 25.65 46

-1.35 1.39 1.24 0.36 0.92 0.16 26.86 46

18 min. EL exposure -0.46 0.59 1.85 55.51 0.12 2.02 64.79 46 48 min. EL exposure -0.56 0.79 1.88 54.06 0.07 2.43 88.63 46 Blink frequency 09:00 – 10:00 EL 18 min. EL exposure -1.71 2.45 2.01 -0.13 1.00 0.01 91.15 46 48 min. EL exposure 0.22 3.45 1.34 34.80 0.21 1.58 70.24 46

0.21 3.42 1.06 5.30 0.54 0.74 57.31 46 Blink frequency 11:30 – 12:30 EL 18 min. EL exposure -0.98 1.61 2.08 0.61 0.86 0.25 45.43 46 48 min. EL exposure 0.08 0.18 1.30 -0.05 1.00 2.1 e-6 50.53 46

-0.60 1.00 0.75 0.01 1.00 5.71 e-5 32.11 46 Blink frequency 14:00 – 15:00 EL 18 min. EL exposure -2.13 0.48 3.34 71.27 0.36 1.09 61.95 46 48 min. EL exposure -2.23 0.63 3.33 77.48 0.21 1.55 76.44 46

-0.22 0.82 2.81 77.01 0.04* 3.06 45.94 46 Blink frequency 16:30 – 17:30 EL 18 min. EL exposure 0.01 2.80 1.30 5.39 0.12 2.02 71.09 46 48 min. EL exposure -3.01 0.61 3.34 75.42 0.27 1.36 90.43 46

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3

Pmin Pmax a B p F SS Df Closing time 09:00 – 10:00 EL 18 min. EL exposure 0.16 0.98 2.64 -18.60 0.34 1.15 78.66 46 48 min. EL exposure -0.72 2.08 1.41 -0.30 0.99 0.04 74.03 46

-2.00 2.04 2.05 0.27 0.94 0.13 41.81 46 Closing time 11:30 – 12:30 EL 18 min. EL exposure -0.93 1.90 1.94 -0.12 1.00 0.01 68.53 46 48 min. EL exposure 0.14 0.91 1.893 -66.33 0.15 1.87 44.45 46

-0.06 0.16 2.83 -53.47 0.92 0.16 34.57 46 Closing time 14:00 – 15:00 EL 18 min. EL exposure 0.16 2.35 3.96 -4.22 0.29 1.28 35.80 46 48 min. EL exposure -1.46 2.19 2.43 -0.44 0.94 0.13 91.83 46

-0.03 3.79 1.31 28.48 0.28 1.31 49.33 46 Closing time 16:30 – 17:30 EL 18 min. EL exposure 0.29 0.87 2.69 -80.03 0.47 0.86 70.98 46 48 min. EL exposure -2.14 2.87 1.38 0.25 0.97 0.09 79.66 46

Total closed time 09:00 – 10:00 EL

18 min. EL exposure 0.12 3.21 3.98 -4.77 0.39 1.03 79.27 46 48 min. EL exposure 0.75 0.87 2.86 -59.07 1.00 0.02 74.13 46

-2.02 2.04 2.17 0.27 0.94 0.13 41.81 46

18 min. EL exposure -0.34 1.41 3.57 -0.20 1.00 0.01 68.53 46 48 min. EL exposure 0.14 0.91 1.89 -65.06 0.15 1.87 44.45 46

-1.33 1.10 1.21 -0.33 1.00 0.08 34.75 46

18 min. EL exposure 0.17 1.93 3.62 -4.81 0.29 1.29 35.79 46 48 min. EL exposure 0.22 3.14 3.38 -61.07 0.79 0.39 90.32 46

-1.81 2.63 1.07 0.36 0.88 0.22 52.79 46

18 min. EL exposure 0.29 0.87 2.68 -76.49 0.44 0.86 70.98 46 48 min. EL exposure -2.14 2.91 1.31 0.24 0.97 0.09 79.66 46

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Pmin Pmax a B p F SS Df Reopening time 09:00 – 10:00 EL 18 min. EL exposure -2.05 1.98 0.54 -0.14 1.00 0.02 82.06 46 48 min. EL exposure -0.70 0.34 0.82 -2.41 0.99 0.05 78.83 46

-0.14 0.86 1.07 5.93 0.97 0.08 41.22 46 Reopening time 11:30 – 12:30 EL 18 min. EL exposure -3.31 2.77 0.51 -0.17 0.97 0.07 67.10 46 48 min. EL exposure -2.26 0.23 0.67 -2.87 0.97 0.08 84.47 46

-0.15 2.67 1.27 27.74 0.95 0.11 65.91 46 Reopening time 14:00 – 15:00 EL 18 min. EL exposure -1.81 1.32 1.44 -0.82 0.48 0.83 35.46 46 48 min. EL exposure -0.38 0.13 0.60 -0.12 1.00 2.26 e-4 92.29 46

0.12 3.66 1.31 47.81 0.95 0.12 78.74 46 Reopening time 16:30 – 17:30 EL 18 min. EL exposure -2.32 0.63 1.07 -2.70 0.44 0.91 80.30 46 48 min. EL exposure -4.65 0.20 1.34 -30.54 0.19 1.67 156.71 46 Pmin Pmax a B p F SS Df

Distal skin temperature 09:00 – 10:00 EL

18 min. EL exposure -0.14 0.23 1.38 0.96 0.32 1.21 0.46 46 48 min. EL exposure -0.28 0.24 3.02 0.58 0.71 0.46 0.84 46

-0.07 -0.02 2.75 78.36 0.79 0.35 1.37 46

-0.21 -0.04 2.29 -67.41 0.20 1.62 3.29 46

-0.46 0.36 1.44 0.26 0.97 0.08 2.68 46

18 min. EL exposure -0.51 0.41 4.17 0.14 0.99 0.03 2.88 46 48 min. EL exposure -0.17 0.32 3.76 -0.38 0.97 0.05 2.99 46

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3

Proximal skin temperature 09:00 – 10:00 EL

-1.05 0.40 3.36 37.66 0.34 1.13 24.56 46

18 min. EL exposure -1.14 2.01 3.01 -0.42 0.84 0.27 28.84 46 48 min. EL exposure -0.28 1.50 1.97 -0.03 1.00 2.45 e-4 52.87 46

-1.63 1.85 1.64 0.19 0.98 0.06 35.22 46

18 min. EL exposure -0.33 0.99 1.20 0.22 1.00 8.52 e-3 42.76 46 48 min. EL exposure 0.19 2.78 1.36 39.63 0.06 2.63 44.18 46

-2.10 0.15 0.87 -2.29 0.447 0.86 26.21 46

18 min. EL exposure -0.38 0.61 0.42 -0.13 1.00 3.65 e-3 17.10 46 48 min. EL exposure -1.36 1.93 2.21 0.15 0.99 0.04 27.45 46 Distal-Proximal gradient 09:00 – 10:00 EL 18 min. EL exposure -0.64 -0.32 2.10 94.59 0.72 0.44 44.55 46 48 min. EL exposure -0.74 2.05 3.60 -16.62 0.58 0.66 52.34 46

-0.42 1.40 3.44 -15.94 0.28 1.30 34.95 46 Distal-Proximal gradient 11:30 – 12:30 EL 18 min. EL exposure -1.09 0.51 2.56 -0.52 0.90 0.19 17.08 46 48 min. EL exposure -1.21 0.38 4.46 -0.33 0.99 0.03 37.45 46

-0.60 0.47 3.61 -0.23 1.00 0.01 23.46 46 Distal-Proximal gradient 14:00 – 15:00 EL 18 min. EL exposure -1.83 -0.20 1.34 -27.83 0.15 1.85 17.91 46 48 min. EL exposure -0.68 -0.08 2.40 -82.79 0.11 2.11 28.43 46

-2.12 2.29 0.71 0.07 1.00 0.01 28.67 46 Distal-Proximal gradient 16:30 – 17:30 EL 18 min. EL exposure -1.49 0.99 2.49 0.14 1.00 0.02 32.67 46 48 min. EL exposure -0.69 2.05 4.60 -4.14 0.72 0.45 62.67 46

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Table S4: Dose response curves were fitted with a modified versio

n of the Naka-Rushton equation:

.

Values of the best fit are shown for all other parameters of ale

rtness, during- or after EL exposure on averaged values over ti

me of day. Significant values are

indicated with *, but none of them reached significance after co

rrecting for multiple comparisons(

α

corrected =0.02). Pmin Pmax a B p F SS Df Sleepiness 18 min. EL exposure -1.82 0.20 3.75 12.96 0.62 0.59 20.42 46 48 min. EL exposure -1.78 0.59 6.08 2.45 0.63 0.58 23.56 46

-0.05 1.79 1.29 10.67 0.03* 3.10 13.47 46 Reaction time 18 min. EL exposure -1.19 -0.02 1.31 -20.50 0.44 0.92 11.87 46 48 min. EL exposure -1.30 -0.08 1.29 -18.65 0.56 0.69 12.82 46

-1.21 -0.07 1.25 -8.23 0.30 1.27 11.84 46 Blink duration 18 min. EL exposure -1.27 0.19 1.22 -16.82 0.93 0.14 24.37 46 48 min. EL exposure -1.68 0.18 1.12 -8.28 0.82 0.29 32.92 46

-1.58 1.74 1.49 0.51 0.71 0.46 28.63 46

10%fastest reaction time

-0.46 -0.05 1.30 -0.17 1.00 0.01 14.74 46

10%slowest reaction time

18 min. EL exposure -0.47 2.05 e-3 1.47 -4.46 0.41 0.96 7.31 46 48 min. EL exposure -1.11 0.64 1.722 -0.66 0.53 0.74 8.13 46

-1.22 -0.02 1.09 -3.55 0.28 1.32 8.89 46

Average reaction time

18 min. EL exposure -1.08 -0.07 1.31 -25.78 0.53 0.74 7.13 46 48 min. EL exposure -1.18 0.59 1.31 -0.56 0.65 0.55 7.80 46

-1.09 -0.10 1.1 -5.51 0.31 1.23 8.03 46

First minute reaction time

-0.94 -0.05 1.36 -36.18 0.16 1.79 7.68 46

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3

Last minute reaction time

-0.69 -0.05 1.33 -26.28 0.70 0.48 7.56 46 Blink frequency 18 min. EL exposure -0.95 1.70 2.08 0.78 0.50 0.80 24.39 46 48 min. EL exposure -1.53 2.11 2.49 0.48 0.78 0.36 38.03 46

-1.22 0.49 3.42 16.38 0.06 2.61 16.71 46 Closing time 18 min. EL exposure 0.30 1.77 3.06 -5.50 0.03* 3.25 24.59 46 48 min. EL exposure -0.43 1.32 2.32 -0.26 0.99 0.03 29.56 46

-1.25 0.20 6.98 1.93 0.89 0.21 21.25 46

Total closed time

-0.03 1.63 1.33 36.98 0.53 0.74 16.58 46 Reopening time 18 min. EL exposure -1.44 1.34 1.43 -0.62 0.66 0.54 23.87 46 48 min. EL exposure -0.20 0.18 2.01 -15.68 0.70 0.47 42.24 46

-0.07 1.76 4.76 -5.82 0.94 0.13 29.39 46

Distal skin temperature

-0.05 0.03 2.59 -164.64 0.61 0.61 1.71 46

Proximal skin temperature

0.18 0.36 2.62 173.30 0.75 0.40 15.14 46

Distal proximal gradient

-0.42 -0.15 2.50 -256.10 0.47 0.86 15.02 46

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