Cihat Ucar (Orcid ID: 0000-0003-3278-7779)

Accepted Article
Tuba Ozgocer (Orcid ID: 0000-0002-4590-1342)

Effects of late-night eating of easily- or slowly-digestible meals on sleep, hypothalamo-pituitary-

adrenal axis, and autonomic nervous system in healthy young males

Running Head: Late-night eating and stress axes

Cihat Uçar1, Tuba Özgöçer2, Sedat Yıldız3*

1
Department of Physiology, Faculty of Medicine, University of Adıyaman, 02000 Adıyaman, ORCID ID:

0000-0003-3278-7779 E-MAIL: ucarcht@gmail.com

2
Department of Physiology, Faculty of Medicine, University of Harran, 63000 Şanlıurfa, ORCID ID:

0000-0002-4590-1342 E-MAIL: tubaozgocer@gmail.com

3
Department of Physiology, Faculty of Medicine, University of Inonu, 44280 Malatya, ORCID ID: 0000-

0002-7872-790X E-MAIL: sedat.yildiz@inonu.edu.tr

*Corresponding author: Sedat YİLDİZ, Department of Physiology Faculty of Medicine University of

İnonu 44280 Malatya/TURKEY, sedat.yildiz@inonu.edu.tr

Key words: Late-night eating, easily-digestible meal, slowly-digestible meal, sleep, HPA, ANS.

Acknowledgements: This study was supported by Inonu University BAP (project #2015-96).

Conflict of ınterest statement: The authors have notified that no competing interests conflict and published

at the stage of preparation of this manuscript.

This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences between
this version and the Version of Record. Please cite this article as doi: 10.1002/smi.3025.

This article is protected by copyright. All rights reserved.

 Effects of late-night eating of easily- or slowly-digestible meals on sleep, hypothalamo-pituitary-

adrenal axis, and autonomic nervous system in healthy young males

Accepted Article
Running Head: Late-night eating and stress axes

Cihat Uçar1, Tuba Özgöçer2, Sedat Yıldız3*

1
Department of Physiology, Faculty of Medicine, University of Adıyaman, 02000 Adıyaman, ORCID

ID: 0000-0003-3278-7779 E-MAIL: ucarcht@gmail.com

2
Department of Physiology, Faculty of Medicine, University of Harran, 63000 Şanlıurfa, ORCID ID:

0000-0002-4590-1342 E-MAIL: tubaozgocer@gmail.com

3
Department of Physiology, Faculty of Medicine, University of Inonu, 44280 Malatya, ORCID ID:

0000-0002-7872-790X E-MAIL: sedat.yildiz@inonu.edu.tr

*Corresponding author: Sedat YİLDİZ, Department of Physiology Faculty of Medicine University

of İnonu 44280 Malatya/TURKEY, sedat.yildiz@inonu.edu.tr

Key words: Late-night eating, easily-digestible meal, slowly-digestible meal, sleep, HPA, ANS.

Acknowledgements: This study was supported by Inonu University BAP (project #2015-96).

Conflict of ınterest statement: The authors have notified that no competing interests conflict and

published at the stage of preparation of this manuscript.

This article is protected by copyright. All rights reserved.

 ABSTRACT

Aim of the current study was to assess the effects of the digestibility of late-night high calorie
Accepted Article
meal on sleep and the activities of the hypothalamo-pituitary-adrenal (HPA) and autonomous
nervous system (ANS) in healthy young males.
For that purpose, effects of an easily-digestible meal (starch + sugar-rich meal, SSR, i.e. dessert)
or a slowly-digestible meal (protein+ fat-rich, PFR, i.e. kebab) were investigated in a crossover
design in 16 participants (20-26 year-old). They did not eat anything after 07:00 pm on Day 0;
had an SSR meal on Day 1 and a PFR meal on Day 2 at 10:00 p.m. HPA and ANS activities
were measured by cortisol awakening response (CAR) and heart rate variability (HRV),
respectively. The participants provided salivary samples for CAR; had a 5-min continuous
electrocardiogram recording for HRV; and filled in sleep questionnaires.
Late-night eating of SSR and PFR diets increased the area under the curve of CAR (p<0.05)
but did not affect HRV parameters (p>0.05). PFR meal significantly disturbed sleep (p<0.05)
The data suggests that increased activity of HPA, but not ANS, might be involved in
pathophysiology of late-night eating and that this might be due to disturbed sleep if slowly –
digestible meal is consumed.

Key words: Late-night eating, easily-digestible meal, slowly-digestible meal, sleep, HPA,
ANS.

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 Introduction
Transition to modern lifestyles has imposed changes on human dietary habits. Specifically,
eating high energy foods late in the night has become a part of everyday life for some people.
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This change in dietary habit is not concordant with biorhythm and reported to be associated
with increased risk for metabolic diseases (Pot, 2017; Nakajima, 2018). For example, late-night
eating has been associated with higher body mass index (Kito et al. 2019), obesity (Kito et al.,
2019; Wang et al., 2014), glucose intolerance (Gu et al., 2020), inflammatory markers
(Martínez-Lozano et al., 2020) and poor cardio-metabolic health (St-Onge et al., 2017; Kutsuma
et al., 2014; Cahill et al., 2013). Late-night eating is classified as eating after 10:00 pm or eating
2 h before sleep at least three times per week (Okada et al., 2019). It might be confused with
night eating syndrome (NES), which is characterized by recurrent episodes of night eating, by
excessive food consumption in the evening or after awakening from sleep (McCuen-Wurst et
al. 2018). Prevalence of late-night eating was between 11-22 % in Japanese women (Okada et
al., 2019). Moreover, about one-quarter of men in young adulthood (aged in their 20s to 40s)
are late-night eaters and they have increased weight gain, type II diabetes and increased
mortality (Kito et al., 2019).

Late-night eating affects sleep parameters (Yeh and Brown, 2014; Lopes et al., 2019) and all of
the above diseases are also co-occurred with sleep impairment (Brown et al., 2017; Okada et
al., 2019). Therefore, it is not known whether cardio-metabolic diseases occur due to direct
metabolic causes associated with late-night eating or whether they manifest as a result of
disturbed sleep caused by late-night eating. Both are possible but probably slowly-digested high
calorie foods (fat and protein) might have greater impact on disrupting the sleep parameters
than that of easily-digested high calorie foods (sugar and starch).

Effects of late night eating on health problems may involve disruption of hypothalamo-
pituitary-adrenal (HPA) axis and the autonomous nervous system (ANS). These are body’s two
main stress axes and might primarily be affected as they are the integral indicators of well-being
(Tsigos et al., 2016). In fact, significant correlations have been found between the end product
of HPA axis, i.e. cortisol secretion, and body mass index (Joseph et al., 2017). Furthermore,
cortisol awakening response (CAR), a measure of the activity of HPA axis as assessed by
salivary cortisol levels immediately following wake up in the morning, has also recently been
associated with sleep parameters (Devine et al., 2017; Uçar et al., 2018), cardio-metabolic
health (Kastaun et al., 2014), obesity (Incollingo Rodriguez AC et al., 2015) and etc. ANS

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 activity, measured non-invasively by heart rate variability (HRV), that is beat-to-beat variation
in heart rate, has been associated with sleep parameters (Tobaldini et al., 2013; Uçar et al.,
2018), cardiac (Haensel et al., 2008) and metabolic problems (Stuckey MI et al., 2014).
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However, it is currently not known whether the detrimental effects of late night eating of high
calorie foodstuffs is manifested by sleep impairment, or disruption of stress axes, namely HPA
and ANS, or both. Moreover, the studies on high calorie foods on sleep parameters should also
include the effects of easily- and slowly-digestible foods as both may have differing impacts on
sleep and associated parameters.

We hypothesize that late-night eating will affect HPA and ANS axes by impairing sleep
duration and quality. As menstrual phases appear to affect both HPA (Wolfram et al., 2011)
and ANS (Bai et al., 2009; Brar et al., 2015), we only included men for this particular
experiment. Moreover, we studied healthy and young participants to observe the magnitude of
changes that are likely to occur before the health is compromised. Aim of the current study was
to assess short-term effects of eating a meal late in the night on sleep quality and on CAR and
HRV parameters in young males in the next morning. In order to get rid of the influence of
inter-individual differences on these parameters, we followed the same individual for three
consecutive days and offered no meal, or high energy meal, or high protein plus fat meal before
sleeping time on days one, two and three, respectively. For that purpose, we chose two types of
diets that are commonly used in Turkey; that are “tulumba tatlısı” made up of crispy fried
doughnuts drenched in sugar syrup (high in sugar and starch) and “Adana kebab” made up of
minced meat with high fat content (high in protein and fat). Normally, they are consumed at
lunch or dinner but for the purpose of the study we requested from the participants to consume
them before sleeping.

2. MATERIALS-METHODS
2.1. Participants
The study was approved by the Malatya Clinical Research Ethics Board (No: 2015/44) and was
conducted with 16 healthy male participants (second year medical students in Inonu University,
Turkey) with an average age of 24 (20-26) years of age and a body mass index of 23.2 (20.9-
26.7). Power analysis was carried out by taking into account the data presented by Stalder et al.
(2010). In the analysis (SPSS), type I error (α) was accepted as 0.05 and type II error (β) was

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 accepted as 0.20. It was calculated that minimum 9 participants was required to carry out the
experiment. Participants did not have chronic diseases and were not using any medication. At
the time of the study, they were apparently healthy and had no complaints about their health.
Accepted Article
The students were in close age range and, as second year students; they were expected to have
same level of anxiety (examination schedules etc.). Study procedure was explained to
participants and they signed voluntary consent forms.

2.2. Procedure
Before starting the study, the participants were met and asked whether or not they had any
health problem (especially digestive system diseases). They were informed about the design of
the study. Then, the informed consent form was signed by them, and the study procedure was
presented in writing. The participants' mobile phone numbers were obtained. Saliva collection
tubes prepared for the study (for three consecutive days), mouthpieces to give saliva samples
and alarm clocks to check the time to give saliva samples were delivered to the participants.
The study was conducted according to "gold standards for cortisol awakening response"
published in 2016 (Stadler et al., 2016). In the night of the first day, the participants did not eat
any food after 19:00. Saliva samples were collected at 0, 15, 30, and 60 minutes after they woke
up in their home in the next morning. The participants filled out surveys in the morning hours.
Then, the questionnaires and the collected saliva samples were brought to the laboratory until
noon in each day. Cortisol is a stable hormone and it has been tested that saliva samples can
stand at room temperature at least for 1-2 days without losing activity (Turpeinen and
Hämäläinen, 2013). Each participant who came to the laboratory was rested for at least half an
hour. A 5-min electrocardiogram (ECG) recording was taken for heart rate variability (HRV)
parameters. On the second day, each participant was given 250 gr of Tulumba dessert (typically
made up of approximately 50 % flour and 50% sugar, fried in vegetable oil) and was said to
consume all of them in the last 1 hour before going bed in the night. Saliva samples were again
collected in the following morning, were brought to the laboratory and ECG record was taken.
On the third day, all participants were taken to a restaurant at 22:00 in the night. All participants
consumed 1 portion (250 g) of high kebab (typically made up of approximately 70 % minced
red meat and 30 % animal fat, grilled). The participants directly went their homes by private
vehicles. These measurements were repeated in the following morning (Figure 1). In the last
two nights they were not allowed to eat or drink anything else (except normal water).
Demographic characteristics of participants are presented in Table 1.

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 Table 1 in here

Figure 1 in here
Accepted Article
2.3. Cortisol awakening response
The participants were given labeled 1.5 ml Eppendorf tubes and adjustable alarm clocks to
collect the saliva samples. Saliva samples were taken on three days of the study immediately
following awakening (0, 15, 30, and 60 minutes after awakening; a total of 12 samples per
person) according to Ozgocer et al. (2017a). Saliva samples were collected by the participants
in their homes. The alarm clocks were adjusted to 15 min (or 30 min for the last sample) after
their first saliva sample is collect immediately after awakening (0 min). In addition, the mobile
phone numbers of the participants were taken and they were sent an informing message at the
preceding nights before each CAR measurement. Saliva samples were taken by using passive
droll method (Granger et al., 2007; Ozgocer et al., 2017a; Ucar et al., 2018). Area under the
curve (ground and increase-AUCg and AUCi, respectively) was calculated (Pruessner et al.,
2003). At the end of saliva collection for CAR, all the samples were taken to laboratory within
an hour and they were kept at -40 C until they were analyzed.

2.4. Measurement of cortisol in saliva

Following thawing, samples were centrifuged at 4000 g for 10 min and the supernatant was
used for ELISA analyses as reported by Ozgocer et al. (2017b). Samples were diluted 5x with
assay buffer and assayed in triplicate.
ELISA procedure: Cortisol-BSA stock solution was diluted with carbonate buffer and added at
200 μL/well to a 96-well microtiter plate. The plate was incubated overnight at +4 °C and
washed 5 times with wash buffer using eight-channel pipette. Some binding sites not occupied
by the coating antigen were then blocked by the blocking buffer (200 μL/well) for 2 h at 37 °C.
Following washing steps (5 times), standard solutions or samples (40 μL/well) and diluted 1st
Ab (antiserum) (40 μL/well) were added in duplicate and incubated at 37 °C for 45 min. After
washing 5 times, biotinylated anti-Rabbit antibody was added (100 μL/ well) and the plate was
incubated at 37 °C for 30 min. The plate was washed 5 times and the streptavidin peroxidase
solution (100 μL/well) was added and the plate incubated for 15 min at +4 °C. Then, the plate
was washed again for 5 times and the substrate solution (150 μL/well) was added and incubated
in dark for 10 min. After incubation, stop solution (50 μL/well) was added and the absorbance

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 was measured at 450 nm using a microplate reader. İntra-assay variation (CV) is % 7.8 and
inter-assay variation is % 7.2. (Ozgocer et al, 2017b).
Accepted Article
2.5. Heart Rate Variability
When the participants brought their saliva samples to the laboratory, they were kept resting for
at least an hour. ECG record of the rested participants was taken for 5 minutes in supine
positions (Uçar et al., 2018). The participants were not allowed to speak, or close their eyes
longer than blinking (they were told not the close their eyes as if they are sleeping but stare at
the ceiling). Poly-Spectrum 8-E was used for electrocardiogram (ECG) recording and heart rate
variability (HRV) analysis was made with the help of a software program (Neurosoft, Russia)
used with the same device. On three consecutive days of the study, ECG recording of each
participant was taken at the same hour.
Time- and frequency-domain parameters of the HRV are determined by the use of software
provided. The most used time-domain variables are SDNN (standard deviation of normal-to-
normal intervals), RMSSD (root mean square of successive differences) and pNN50 (normal-
to-normal R-R intervals that differ by more than 50 ms) parameters. The most used frequency-
domain variables are TP (total power), LF (low frequency), HF (high frequency), LF n.u. (low
frequency normalized units), HF n.u. (high frequency normalized units) and LF/HF ratio (Task
Force., 1996).

2.6. Questionnaires
Karolinska Sleep Diary, Karolinska Sleep Questionnaire and Pittsburgh Sleep Quality index
were used.
2.6.1 Karolinska Sleep Diary
It was used to get information about the person’s sleep in preceding night. The sleep diary used
the 7 question version of the scale as reported Hansen et al. (2012). Disturbed sleep score was
calculated as the average of the answers given to four questions: the questions (1) ‘Was it
difficult for you to fall asleep?’, (2) ‘Were you uneasy in your sleep? (Did you keep turning
around)’, (3) ‘Did you wake up very early and no get back to sleep again?’, (4) ‘How many
times did you wake up at night?’ Disturbed sleep score was calculated as the average of the
answers given to four questions: the questions were at the scale of 7 as 0, 1, 2, 3, 4, 5, too much.
A high score meant more disturbed sleep. Awakening problems score was calculated as the
average of the answers given to three questions: the questions (1) ‘How was your sleep?’, (2)
‘How rested do you feel?’, (3) ‘Was it easy for you to wake up?’ were at the scale of 7 as, 0, 1,

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 2, 3, 4, 5, too much, high score meant more awakening problems. Duration of sleep was
assessed with the answers given to questions ‘What time did you go to bed?’ and ‘What time
did you wake up?’.
Accepted Article
2.6.2. Karolinska Sleep Questionnaire
Karolinska Sleep Questionnaire, which includes seven questions, gives information about a
person’s sleep in the last four weeks. The answers given to questions are marked in a scale of
five and the averages of these are taken (1: Always, 5: Never). Sleep disturbance was calculated
with the average of the answers given to four questions: (1) ‘How frequently was your sleep
insufficient and you had sleep impairment?’, (2) ‘How frequently did you have problems to fall
asleep?’, (3) ‘How frequently did you wake up very early and could not get back to sleep
again?’, (4) ‘How frequently did you wake up again and again and could not go back to sleep?’,
a low score meant more sleep disturbed. The awakening problems score (range 1—5) were
calculated as the mean score of two items which assessed: (1) ‘How often did you have
difficulties waking up?’, (2) ‘How often did you feel exhausted when you woke up?’. In
addition, one item measured the overall sleep quality: ‘How do you rate your overall quality of
sleep?’ with 5 response categories ranging from 1 = excellent to 5 = poor.

2.6.3. Pittsburgh Sleep Quality Index

The information about the sleep quality of the participants in the last month was assessed with
Pittsburgh Sleep Quality Index.

2. 7. Statistical Analyses
IBM SPSS Statistics program was used in analyses. Shapiro-Wilk test was used to find out if
the data were normally distributed. Data had non-normal distribution and therefore Friedman
test was used to find out effects of diets on cortisol, HRV, and sleep parameters. In Friedman’s
test, the type of food offered was used as independent variable and cortisol, HRV, and sleep
parameters were used as dependent variables. Wilcoxon rank test was used for post hoc paired
comparisons. The correlations were calculated with Spearman Rho coefficient. The data were
presented as median and minimum and maximum values. P<0.05 was considered as statistically
significant.

Results

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 Effect of late night eating on sleep parameters
Table 2 shows the sleep parameters in the same night in participant and Table 3 shows the sleep
Accepted Article
parameters in the for past 4 week in participants who did not eat anything (control) before
sleeping or who ate SSR meal (dessert) or PFR meal (kebab) before sleeping. The deterioration
of sleep parameters was found to be significantly higher at the night when the PFR meal was
eaten compared to the control day (p<0.05). However, there was no significant difference
between the groups in terms of awakening problems and duration of sleep.

Table 2 in here

Table 3 in here

The effect of late-night eating on cortisol awakening response

Figure 2 and Table 4 show the CAR observed in the next morning in individuals who did not
eat anything (control) before sleeping and who ate SSR meal (dessert) or PFR meal (kebab)
before sleeping. Eating a meal (dessert and food) before sleeping increased the AUCİ (Figure
3) compared to the control group (no meal) rather than the type of food eaten before sleeping
(p<0.05) and the cortisol concentration measured 15 minutes following wake-up in the morning
after eating dessert was significantly increased compared to the control day.

Figure 2 and 3 in here

Table 4 in here

The effect of late-night eating on HRV parameters

Table 5 shows the HRV parameters obtained in the following morning in individuals who did
not eat anything (control) before sleeping and who ate SSR meal (dessert) or PFR meal (kebab)
before sleeping. There was no statistically significant difference between the groups in terms
of HRV parameters.

Table 5 in here

Correlations between Sleep Parameters and Heart Rate Variability Parameters

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 Awakening problems were negatively and significantly correlated with SDNN, RMSSD,
pNN50 and HF (Table 6). Sleep duration on the other hand was positively and significantly
Accepted Article
correlated with SDNN, RMSSD, pNN50, LF and HF (Table 6). Correlations between AUCg –
AUCi and sleep parameters showed in Table 7. AUCg was significantly correlated with LF n.u
(R-square = 0.455; P<0.05), HF n.u (R-square = - 0.455; P<0.05) and LF/HF (R-square =
0.456; P<0.05). AUCi was significantly correlated with LF n.u (R-square = 0.419; P<0.05),
HF n.u (R-square = - 0.419; P<0.05) and LF/HF (R-square = 0.419; P<0.05).

Table 6 in here

Table 7 in here

4. DISCUSSION
The current study shows that eating a high SSR meal or high PFR meal late in the night
disturbs sleep parameters, increases cortisol awakening response but does not change
sympathovagal balance in the next morning in young males. Although everyone is familiar with
disturbing effects of consuming diets late in the night on sleep parameters; findings of the
current study appear to be beyond this, suggesting that late night slowly-digestible meal
consumption increases the activity of HPA, but not ANS, in the next morning. Findings also
support our hypothesis that metabolic problems associated with late night eating might include
immediate changes on HPA axis.

Sleep is disturbed if a diet rich in protein+fat is consumed late in the night

In the present study, disturbed sleep was observed in participants consuming slowly-
digestible PFR meal rather than those consuming easily-digestible SSR meal. A negative
correlation between sleep-onset time and eating fatty food approximately 1 hour before sleeping
has also been reported in young healthy individuals (Crispim et al., 2011). In the current study,
relationships between sleeping pattern and food intake have been examined in healthy subjects.
It seems that eating late only one night has similar effects observed in people who are classified
late night eaters or nigh eating syndrome (Gallant et al., 2012). Taken all together, the results
obtained from the present study and that of others, slowly-digestible PFR meal seem to disturb
sleeping more than easily-digestible SSR meal.

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 Late night eating increases CAR irrespective of meal type
In the present study, it was found that eating late at night affected CAR regardless of the
type of meal eaten. To the best of our knowledge, this is the first study investigating the effect
Accepted Article
of late night eating on CAR in the next morning. However, there are studies examining the
correlations between meal time, type of food and cortisol. Post-prandial cortisol concentrations
were either decreased (blood samples of 8 female participants, Racz et al., 2015) or increased
(blood samples of 8 participants, Stimson et al., 2014). It has been reported that blood cortisol
reflects total cortisol levels whereas salivary cortisol represents free (active) cortisol (Gatti et
al., 2009; Kudielka et al., 2009). In another study, 22 female participants were given diets at
different lunch times [early lunch group: 13:30, late lunch group: 16:30, dinner time: 20:00 (it
was the same for both groups)] for 6 days, and the diurnal rhythm of cortisol was examined on
the next day (Bandin et al., 2015). The salivary cortisol concentration approximately 1 hour
after awakening in the morning and the diurnal cortisol concentration were found to be lower
in the late lunch group than the early lunch group. Another study focused on the type of food
(vegetable or protein-containing food) and found no effect on salivary cortisol concentration,
except a post-prandial increase (Garde et al., 2009). The study of Nonino-Borges et al,. (2007)
focused on offering total daily intake (1000 kcal/d) once in the day either at 09:00-11:00 h or
18:00-20:00 h in 12 obese women and found no change in diurnal salivary cortisol levels taken
6 times per day. In our study, we took 4 saliva samples during awakening and probably, this
made awakening cortisol more robust. Thus, late night eating appears to increase awakening
cortisol concentration although available literature suggests post-prandial change without
diurnal variation.
Late-night eating has been reported to cause many metabolic disorders. For example,
late-night eating has been associated with atrial fibrillation (Nakajima et al., 2014), with
hyperglycemia (Nakajima et al., 2015), obesity (Kito et al.,2019), impaired sleep (Yeh and
Brown, 2014; Lopes et al., 2019), and delayed sleep onset (Sato et al., 2011). In the present
study, it was found that eating high PFR foods before sleeping disturbed night sleep but SSR
foods did not. It is known that sleep disturbances affect CAR (Hansen et al., 2012), and thus,
increased awakening cortisol might be indirect consequence of disturbed sleep rather than being
direct effect of the diet. The exact role of CAR is not known (Law et al., 2015) but it has been
associated with many metabolic diseases (Kuehl et al., 2015; Bengtsson et al,. 2010) and
therefore, future studies should focus on diet type, meal time, dietary habits and their metabolic
consequences, especially those who develop metabolic diseases. Moreover, salivary cortisol
seems to be an easy and non-invasive tool to study these interactions.

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 Late night eating and type of meal did not affect HRV
As HRV is associated with impaired sleep (Tobaldini et al., 2013; Uçar et al., 2018) and
with cardiac (Haensel et al., 2008) and metabolic problems (Stuckey MI et al., 2014), we were
Accepted Article
expecting to observe a change in HRV parameters in high calorie meal groups. However, it was
found that late night eating did not have any effect on heart rate variability (HRV) parameters
on the next day. In line with this, in a study involving 10 healthy participants, it was reported
that late night eating did not have any effect on autonomic nervous system (Sato et al., 2011).
In the present study, significant correlations were found between HRV parameters and sleep
parameters and cortisol measured after wake-up in the morning. The SDNN, RMSSD and
pNN50 parameters were found to be positively associated with sleep duration but negatively
associated with awakening problems. It is likely that decreased sleep duration and wake-up
issues lead to a stress in individuals and negatively affect their daily activities. The cortisol
concentrations measured AUCg and AUCi after wake-up were positively correlated with LF/HF
ratio and LF norm and negatively correlated with HF norm. The fact that both LF/HF ratio and
LF n.u are high and HF n.u is low indicates that sympathovagal activity is high in favor of the
sympathetic system (Shaffer et al., 2014); therefore this shows that the sympathetic system is
also activated to some degree especially in participants with a high cortisol level.

Strength and limitations of the study

In a crossover design, this study investigated the effects of late night diets on three
consecutive days in the same participants. Use of paired comparisons for the same participants
throughout the study in a repeated design measurement appears to overcome the possible wide
inter-individual variation observed in CAR, and in a lesser extent in HRV, parameters. Thus
each participant served as his own control and this strengthened the power of the statistical
comparison. Additionally, the participants were enthusiastic medical students or post-graduate
students and therefore this also helped smooth progression of the study. The first author took
necessary measures to make sure that they complied with the methodology. However, salivary
samples were collected at home but they were informed about the importance with compliance.
In fact, proper compliance was further evidenced by cortisol awakening response which is
similar to the literature. Although they were enthusiastic medical students and we are satisfied
with compliance; this can be accepted as a limitation of the study. Another limitation of the
study was the study population. We investigated healthy males and it might be difficult to
generalize the results to the whole population. Therefore, future studies should focus on
overweight and unhealthy populations to investigate the changes occurred in sleep, CAR and

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 HRV. Moreover, new studies are required to study these parameters in women by giving special
emphasis menstrual age and phases.
Accepted Article
5. CONCLUSION
In conclusion, eating a high calorie slowly-digestible PFR meal late in the night caused
sleep disturbances; whereas eating both SSR meal and PFR meal increased cortisol awakening
response without affecting autonomic balance. The results indicate that disturbed sleep and
HPA axis activity may play important roles in the pathophysiology of diseases caused by late
night eating. In this context, salivary cortisol measurement appears to be a sensitive parameter
for these types of studies.

6. Acknowledgement
This study was supported by İnonu University BAP (project #2015-96).

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Figure and Table Captures

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 Figure 1. Study procedure. Sixteen male young participants did not eat anything late in the first
night of the study and they were allowed to have their normal sleep. In the next morning, they
took their salivary samples for cortisol awakening response (CAR) at 0, 15, 30 and 60 min post-
Accepted Article
awakening, had 5 min electrocardiogram recording for heart rate variability (HRV), filled in
questionairres about their sleep duration and quality and had their blood pressure (BP)
measured. The same procedures were repeated for the second and third days except that in the
second and third days they were offered high SSR meal or high PFR meal, respectively within
one hour before going bed.

Figure 2. Cortisol awakening response (CAR, ng/ml) observed in the following morning in
subjects who do not eat anything before sleep (control) or consume SSR meal or PFR meal.
Values represent median data.

Figure 3. Area under the curve increase (AUCi, min.ng/ml) was higher in SSR meal and PFR
meals than the control group (P<0.05). Cortisol levels with different letters are statistically
different from each other.

Table 1. General characteristics of participants (n = 16).

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 Participants %
Age 20-26 years
Gender
Accepted Article
Male (n) 16 (male) 100
Occupation
College student 14 87.5
Graduate student 2 12.5
Smoking status
Smoker 2 12.5
Non-smoker 14 87.5

Table 2. The sleep parameters obtained in the same night in individuals who did not eat
anything (control) before sleeping and who ate SSR meal (dessert) or PFR meal (kebab) before

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 sleeping. The Karolinska Sleep Diary is scored between 1-5 points and high score indicates
high sleep disturbances. Disturbed sleep score with different letters are statistically different
from each other.
Accepted Article
GROUPS
Parameters Control SSR meal PFR meal

Disturbed Sleep 1.0 (0.8-1.8)a 1.0 (0.8-2.5)ab 1.8 (0.8-2.8)b

Awakening problems 2.0 (1.3-3.6) 2.3 (1.3-3.3) 2.3 (1.0-3.6)
Sleep Duration (h) 6.1 (4.5-9.0) 6.2 (3.0-9.0) 6.5 (4.0-9.5)

Table 3: The sleep parameters obtained in the past four week in participants who did not eat
anything (control) before sleeping and who ate SSR meal (dessert) or PFR meal (kebab) before
sleeping. The Karolinska Sleep Questionnaire is scored between 1-5 points and low score

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 indicates high sleep disturbances. If the Pitsburgh Sleep Quality Index (PSQI) score is > 5, it
indicates poor sleep.
GROUPS
Accepted Article
Parameters Control SSR meal PFR meal

Disturbed Sleep 4.25 (2.75-5) 4.25 (2.75-5) 4.25 (2.75-5)

Awakening problems 3.5 (1-4.5) 3.5 (1-4.5) 3.5 (1-4.5)
Sleep Quality 3 (1-4) 3 (1-4) 3 (1-4)
PSQI Score 4 (1-8) 4 (1-8) 4 (1-8)

Table 4. Cortisol awakening response (CAR, ng/ml) and area under the curve (AUC,
min.ng/ml) values in the next morning in the participants who did not eat anything (control)
before sleeping and who ate SSR meal (dessert) or PFR meal (kebab) before sleeping. All values

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 are presented as median (min-max). Cortisol levels with different letters are statistically
different from each other.
Accepted Article
GROUPS
Parameters Control SSR meal PFR meal
CAR
0 min (awakening) 19.4 (1.97-221) 21.1 (9.04-190) 17.2 (2.88-115)
+15 min 21.2 (8.64-80)a 27.9 (7.55-116)b 29 (8.96-103)ab
+30 min 32.3 (11.3-168) 37.1 (8.13-477) 42.3 (7.76-137)
+60 min 24.8 (6.35-151) 26.9 (4.74-2099) 25.7 (4.58-168)
AUCg 1499 (573-6919) 1884 (434-44877) 1719 (534-5291)
AUCi 565 (193-4524)a 1057 (151-40077)b 886 (115-3190)b

Table 5. Heart rate variability (HRV) parameters in the next morning in the participants who
did not eat anything (control) before sleeping and who ate SSR meal (dessert) or PFR meal
(kebab) before sleeping. Values represent median (min-max). There was no statisticaly
significant difference between the groups.

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 GROUPS
HRV Parameters Control SSR meal PFR meal
Heart Rate (bpm) 69 (56-89) 68 (57-86) 68 (53-101)
Accepted Article
SDNN (ms) 60 (31-123) 63 (40-105) 53 (24-98)
RMSSD (ms) 57 (16-135) 51 (23-157) 49 (11-118)
pNN50 (%) 17.5 (0.3-65) 14.8 (1-83) 15.3 (0.2-64)
VLF (ms2) 1287 (341-3751) 1268 (403-3744) 1178 (193-2384)
LF (ms2) 953 (186-3973) 895 (248-4227 660 (191-4060)
HF (ms2) 1154 (81-7848) 1060 (138-6815) 670 (72-5454)
LF n.u 53 (21-78) 56 (9-82) 53 (16-78)
HF n.u 47 (21-78) 43 (17-90) 46 (21-83)
LF/HF oranı 1.13 (0.28-3.71) 1.29 (0.1-4.81) 1.16 (0.2-3.71)
% VLF 31.6 (15.5-73.3) 35.2 (13.1-70) 33.6 (8.7-59.4)
% LF 28.3 (13.5-61.9) 24.2 (8.1-48.7) 30.6 (11.4-61.7)
% HF 23.9 (9.3-62.7) 25.4 (7.2-78.8) 27.9 (12.9-63.9)

Table 6: Correlations between sleep and HRV parameters (in each cell, upper value is R-
squared, lower values is p). Significant correlations are highlighted.
Parameters SDNN RMSSD pNN50 LF HF LF/HF

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 Awakening -0.489 -0.456 -0.377 -0.267 -0.446 0.285
problems 0.001 <0.05 <0.05 0.080 <0.05 0.061
Accepted Article Sleep 0.310 0.349 0.371 0.311 0.361 -0.224
duration <0.05 <0.05 <0.05 <0.05 <0.05 >0.05

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 Accepted Article
Table 7: Correlations between AUCg –AUCi and sleep parameters (in each cell, upper value is R-squared, lower values is p). Significant correlations
are highlighted.
Karolinska Sleep Questionnaire (past four
Karolinska Sleep Diary ( same day)
( past four week) week)
Parameters Disturbed Awakening Sleep Duration Disturbed Sleep Awakening Sleep Quality PSQI Score
Sleep Problems Problems
AUCg 0.290 0.247 0.150 0.098 -0.077 0.062 0.033
<0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
AUCi 0.273 0.118 0.196 0.230 0.064 -0.154 -0.069
0.055 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05

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Accepted Article

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Accepted Article

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Accepted Article

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