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Resilience of the Immune System in Healthy

Young Students to 30-Hour Sleep Deprivation
with Psychological Stress

Article in NeuroImmunoModulation · April 2013

DOI: 10.1159/000348698 · Source: PubMed

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 Original Paper
Neuroimmunomodulation 2013;20:194–204
DOI: 10.1159/000348698
Resilience of the Immune System in Healthy
Young Students to 30-Hour Sleep Deprivation
with Psychological Stress
Pini Matzner a Ofir Hazut a Reut Naim a Lee Shaashua a Liat Sorski a
Ben Levi a Avi Sadeh a Ilan Wald a Yair Bar-Haim a, b Shamgar Ben-Eliyahu a, b
a
School of Psychological Sciences and b Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
Key Words
Cytokines · Humans · Immune resilience · Immunity ·
Natural killer cells · Resilience · Sleep deprivation · Stress
Abstract
Objective: Young adults often encounter sleep deprivation
and stressful events. Both have been separately reported to
modulate immunity, and occasionally they occur simultane-
ously. We assessed the combined effects of these condi-
tions on immune competence in healthy students. Meth-
ods: Twenty-three participants (mean age 24 years; SD 1.86;
14 females) were exposed to 30 h of sleep deprivation during
which they conducted physiological, social and cognitive
tasks. The control group consisted of 18 participants (mean
age 23.67 years; SD 1.46; 11 females). All participants under-
went cognitive and psychological evaluations at 10:00 AM,
followed by blood and saliva collection, 3 days before sleep
deprivation induction and on the morning following it. Im-
mune/endocrine measures included blood counts of lym-
phocytes, granulocytes, monocytes and natural killer (NK)
cells; levels of several cell surface markers; NK cytotoxicity;
plasma levels of interleukin (IL)-6, IL-10, dehydroepiandros-
terone and neuropeptide Y, and plasma and salivary cortisol
levels. Results: Although the experimental protocol signifi-
cantly elevated state anxiety and psychological dissociation
© 2013 S. Karger AG, Basel
1021–7401/13/0204–0194$38.00/0
E-Mail karger@karger.com
www.karger.com/nim
Received: November 15, 2012
Accepted after revision: January 30, 2013
Published online: April 27, 2013
levels, no effects were evident in any of the immunological/
endocrine indices. In contrast, expected sex differences in
immune measures were found, including significantly high-
er NK cytotoxicity and monocyte counts in males, validating
the integrity of the measurements. Conclusions: The find-
ings suggest resilience of the immune system to a combined
sleep deprivation and stressful exposure in young adults,
while previous studies reported immune perturbations fol-
lowing either of these conditions separately. These apparent
contradictions might reflect differences in the study design
or in the methodology used for immunological assessments,
including the time of sample collection, the combination of
sleep deprivation with stress and our in vivo assessment of
cytokine levels. Copyright © 2013 S. Karger AG, Basel
Introduction
The lifestyle of adolescents and young people often
involves extensive deprivation of night sleep. Addition-
ally, epidemiological reports indicate that approximately
one third of the general population in the United States
exhibits at least one symptom of insomnia [1]. Thus,
studying the potential impacts of sleep deprivation on
Tel-Aviv University Lib. of Life Science and Med.
health outcome is important.
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Prof. Shamgar Ben-Eliyahu
School of Psychological Sciences
Tel Aviv University
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Tel Aviv 69978 (Israel)
E-Mail shamgar @ post.tau.ac.il
 Sleep is a critical biological process for normal func-
tioning of all known mammals [2, 3] and has an impor-
tant role in learning and memory consolidation [4, 5],
regulation of hormonal levels [6] and immune activities
[7]. Sleep and its associated circadian rhythms are hy-
pothesized to fulfill a restorative role, preventing accu-
mulated perceptual overload of the cerebral cortex [8],
and ensuring stable and healthy cytokine rhythms [9].
Sleep and immunity have been suggested to mutually
affect each other. Specifically, sleep deprivation has been
associated with a tendency towards a proinflammatory
state, as reflected by a higher potential of monocytes to
produce interleukin (IL)-6 and tumor necrosis factor-α
upon in vitro activation [10, 11]. Sleep deprivation was
also reported to modulate natural killer (NK) cell cyto-
toxicity, with some studies showing reduction and some
enhancement in their activity [9, 12, 13]. Additionally,
human systemic levels of prolactin, estradiol and cortisol
were found to correlate with total sleep latency, REM
sleep latency and the percentage of slow-wave sleep fol-
lowing either total sleep deprivation or selective REM de-
privation [14]. Inversely, the impact of immunity on
sleep has long been examined, and many cytokines were
found to regulate sleep patterns. For example, proinflam-
matory cytokines, including IL-1, are known to be elevat-
ed in response to infections [15], and both intracerebro-
ventricular and peripheral injections of proinflammatory
cytokines were shown to impact sleeping patterns [16,
17]. Similarly, humans subjected to a viral infection re-
ported a growing desire to sleep [18]. Indeed, some stud-
ies suggest that proinflammatory cytokines increase
slow-wave sleep and sleep duration, while anti-inflam-
matory cytokines inhibit sleep [19–21]. However, other
studies suggest a more complex association between
sleep and immunity [22–24].
Immunity was repeatedly shown to be affected by psy-
chological and physiological stressors through various
mechanisms. Some investigators suggested a dichotomy
between the impact of acute and chronic stress, namely
that chronic stress suppresses immunity and reduces re-
sistance to diseases, while acute stress enhances immu-
nity and disease resistance [25, 26]. However, several
acute stressors were actually shown to suppress various
immune indices, including peripheral levels of NK acti-
vity and Th1 cytokine levels [27–30]. Acute stress was also
shown to increase proinflammatory cytokines, including
IL-6 [31].
Overall, it appears that immunity, stress and sleep are
mutually interacting processes, and that sleep deprivation
and stress can often lead to similar immune consequences,
Immune Resilience in Students following
Stress and Sleep Deprivation
potentially through common proinflammatory responses.
Indeed, some psychological and health disorders, includ-
ing depression and cardiovascular diseases, seem to in-
volve irregularities in two or three of these processes [32,
33], suggesting the biological and clinical significance of
these interactions. Importantly, under naturalistic cir-
cumstances, sleep deprivation is often accompanied by
stress or continuous activity. For example, students study-
ing for examinations, professionals in demanding jobs,
parents to newborns or soldiers during active service
may all experience sleep deprivation alongside maintain-
ing activity and/or being subjected to stressful circum-
stances. Thus, in order to simulate such circumstances
and measure their impact on the immune system, we em-
ployed a research protocol combining sleep deprivation
with potentially stressful activities.
We compared young male and female students sub-
jected to a 30-hour sleep deprivation alongside psycho-
logical stress, with a control group of participants. Spe-
cifically, we assessed multiple immune, endocrine, cog-
nitive and psychological indices within participants
before and after the sleep deprivation protocol. The bio-
logical indices studied were those previously reported to
be affected by either stress or sleep deprivation [14, 34–
36], as we herein assess the combined effect of these con-
ditions on immunity. The psychological and cognitive
outcomes of this study will be described in detail else-
where, while this paper focuses on the immunological
and endocrine outcomes of the sleep deprivation and
stress paradigm.
Materials and Methods
Participants
Forty-one young healthy undergraduate psychology students
(mean age = 24 years; SD = 1.86; 25 females) chose to participate
in the study after being briefly informed about the paradigm. Par-
ticipants were randomly assigned into a stress group (23 partici-
pants, mean age 24 years; SD 1.86; 14 females) and a control group
(18 participants, mean age 23.67; SD 1.46; 11 females). Study par-
ticipation fulfilled students’ requirements for participating in ex-
periments. The study was conducted in 2 replicates (4 weeks
apart), each including about half of the participants in each group.
The study was approved by the Institutional Review Board, and all
participants provided a written informed consent.
Experimental Design and Specific Timelines
The experimental design is illustrated in figure 1. All parti-
cipants first reported to the laboratory on Monday morning
(09:00 AM), 3 days before the planned sleep deprivation night, to
conduct a battery of psychological tests and to provide baseline
saliva and blood samples. Participants were then randomly as-
Tel-Aviv University Lib. of Life Science and Med.
signed to either the stress or the control group. Three days later,
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Neuroimmunomodulation 2013;20:194–204 195
DOI: 10.1159/000348698
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 Control group Stress group

Blood and saliva Blood and saliva

09:00– Mon 09:00–
collection, actigraphy collection, actigraphy
11:00 AM 11:00 AM
distribution distribution

Thu Early wake-up:

04:00 AM beginning of sleep
deprivation period

09:00 PM Reporting to the lab

Normal daily routine

Fri
09:15 PM Psychological tests,
to cognitive and social
06:30 AM stress tasks

Blood and saliva Blood and saliva

09:00– 09:00–
collection, actigraphy collection, actigraphy
11:00 AM 11:00 AM
collection collection

Fig. 1. Participants from both groups first reported to the labora-
tory on Monday morning (09:00 AM) to conduct a battery of psy-
chological tests followed by the collection of saliva and blood sam-
ples. Three days later (Thursday), participants from the sleep-de-
prived group were awakened at 04: 00 AM and reported to the
laboratory at 09:00 PM. Batteries of psychological tests, and cogni-
tive and social stress tasks were employed throughout the night
period. Participants of the control group reported to the labora-
tory on Friday 09:00 AM, and both groups underwent a final bat-
tery of psychological tests, and provided saliva and blood samples.

on the day of the experiment (Thursday), participants of the stress
group were awakened at 04:00 AM and allowed to maintain their
daily routine, while continuously being monitored for staying
awake (by wearing an Actigraph, Mini Motionlogger, Ambula-
tory Monitoring Inc. and scheduled phone calls). At 09: 00 PM,
these participants reported to the laboratory and were fully active
throughout the night until 10:00 AM Friday morning. At 4 time
points during the night [(i) 09:15–11:25 PM, and (ii) 00:00–02:25,
(iii) 02:40–06:00 and (iv) 06:30–08:25 AM], different batteries of
psychological tests and social stress tasks were employed (see be-
low). Participants of the control group reported to the laboratory
on Friday 09:00 AM, when participants of both the stress and the
control groups underwent a final battery of psychological tests,
and provided the second set of saliva and blood samples. The time
points of saliva and blood collection were counterbalanced be-
tween groups and maintained constant per participant for the
2 collection days in each replicate.
The following composition of tests and tasks were given
scale, continuous performance test and text presentation task; (ii)
analog mood scale, dot probe while putting the nondominant
hand in ice water and continuous performance test; (iii) arithme-
tic task, analog mood scale and social stress task, and (iv) group
text presentation.
Saliva Collection
Saliva was collected using Salivatte swabs (Sarstedt, Germany)
that were chewed for 60 s and placed back in the saliva tube. To
overcome potential circadian-related cortisol alterations, the ex-
act timing of saliva collection from each participant was matched
along the 2 collection days and counterbalanced between groups.
Salivattes were then centrifuged at 1,000 g for 2 min, and the ex-
tract was collected and stored at –32 ° C for later analyses.
Blood Collection
Ten milliliter of blood were drawn by a certified nurse from
the cubital vein into a heparinized syringe (50 units/ml) and
Tel-Aviv University Lib. of Life Science and Med.

through the nightly time points indicated above: (i) analog mood were kept at room temperature for a maximum of 2 h before be-
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DOI: 10.1159/000348698 Levi/Sadeh/Wald/Bar-Haim/Ben-Eliyahu
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ing processed. Blood samples were drawn following the saliva
collection, between 09: 00–11: 00 AM, and the exact timing of
blood sampling from each participant was also matched along
the 2 collection days and counterbalanced between groups (to
overcome potential circadian-related hormonal and cytokine
alterations). A blood aliquot from each sample was centrifuged
for 20 min at 931 g at 4 ° C, and plasma was collected and stored
at –32 ° C.
Assessment of NK Cytotoxicity
Radiolabeling of K562 Target Cells. Standard NK-sensitive
[37] K562 erythromyeloid tumor cells were used as targets in the
NK cytotoxicity assay. Cells were grown in complete medium
[CM; RPMI 1640 medium supplemented with 10% heat-inacti-
vated fetal calf serum, 50 μg/ml of gentamycin, 2 mM of L-gluta-
mine, 0.1 mM of nonessential amino acids and 1 mM sodium
pyruvate (Biological Industries, Beit Haemek, Israel)] at 37 ° C
in 100% humidity and 5% CO2. For radiolabeling, 4 × 107 cells
were incubated for 1.5 h with 200 μCi of 51Cr, 200 μl of fetal calf
serum and 150 μl of CM. After incubation, cells were washed
3 times with CM (300 g, for 10 min) and adjusted to the desirable
concentrations.
NK Cytotoxicity Assay. The cytotoxic activity of NK cells was
assessed using a standard whole-blood 4-hour 51Cr release assay.
This procedure assesses antitumor cytotoxicity of NK cells per
milliliter of blood without removing any cell subtypes or serum
content. Eight aliquots of 200 μl of heparinized whole blood
were placed in a microtiter plate. To achieve 8 different effector-
to-target (E:T) ratios, 2 × 105 radiolabeled K562 cells/well, in
50 μl of CM, were added on top of the blood aliquot for the low-
est E:T ratio, and half of these target cells (in 50 μl of CM, using
serial dilutions) were used for each additional higher E:T ratio.
To determine spontaneous and maximal 51Cr release, whole
blood was substituted with CM or Triton X-100 (Sigma, Rehov-
ot, Israel), respectively. Plates were centrifuged at 500 g for
10 min and were then incubated for 4 h in 100% humidity, 5%
CO2 at 37 ° C. Following incubation, plates were centrifuged
again at 500 g for 10 min at 4 ° C, and 100 μl of the supernatant
were recovered from each well for assessment of radioactivity in
a γ-counter in order to calculate specific target cell lysis. Earlier
studies indicated that cytotoxicity measured using this proce-
dure is attributable to NK cells rather than to other cell types or
soluble factors [38]. Cytotoxicity was calculated as (sample re-
lease × correction – spontaneous release)/(total release – spon-
taneous release), where correction is the percentage of the super-
natant within the volume of the well (i.e. excluding the hemato-
crit volume). This correction is necessary because the 51Cr
released by the labeled target cells is found only in the superna-
tant above the red blood cells.
Flow Cytometry
FACS analysis was used to assess the number of leukocyte sub-
populations (including lymphocytes, NK cells, monocytes and
granulocytes), as well as expression levels of different activation
markers (see below).
Sample Preparation. A 50-μl aliquot of whole blood was added
to 10 μl of PBS supplemented with 2% FCS and 0.1% NaN3
(PBS++) containing a full set of conjugated antibodies for identi-
fication. Samples were kept in the dark at room temperature
thereafter. Following a 15-min incubation period, 1 ml of FACS
Immune Resilience in Students following
Stress and Sleep Deprivation
lysing solution (Becton Dickinson) was added, and 12 min later,
samples were centrifuged at 670 g for 5 min and the lysate was
aspirated. Cells were rewashed twice with 1 ml PBS++ (5 min
centrifugation, 670 g) and resuspended in 500 μl of PBS++ for
flow cytometry using a FACScan (Becton Dickinson).
Identification of Leukocyte Subsets. Lymphocytes, granulo-
cytes and monocytes were identified based on forward and side
scatters. Within the lymphocytes, NK cells were identified as
CD3– (using PE-Cy5-conjugated anti-human-CD3, Biolegend)
and CD16+ (APC-Cy7-conjugated anti-human-CD16, Bioleg-
end) and/or CD56+ (PE-conjugated anti-human-CD56, Serotec).
Evaluating Cell Marker Expression Levels. Expression levels of
the following activation markers were assessed on relevant cell
types: HLA-DR (FITC-conjugated anti-human-HLA-DR, Bioleg-
end), NKG2D (APC-conjugated anti-human-CD314, Serotec),
Fas ligand (FITC-conjugated anti-human-CD178, Serotec) and
CD11a (APC-conjugated anti-human-CD11a, Serotec).
Evaluating Leukocyte Numbers. To assess the absolute num-
bers of cells per microliter of blood (for each specific leukocyte
subset and for the total number of leukocytes), we added 300
polystyrene microbeads (20 μm; Duke Scientific, Palo Alto, Ca-
lif., USA) per microliter of sample studied. Following cytometry,
the formula: CD3+ (n) / microbeads (n) × 300 was used to calcu-
late the number of CD3+ (T cells) per microliter of sample. The
same formula was used for each other leukocyte subset and for
the total number of leukocytes. The coefficient of variation for
this method was found in our laboratory to be 6% for identical
samples [27, 39, 40].
Assessment of Cytokines, Cortisol, Dehydroepiandrosterone
and Neuropeptide Y
Based on manufacturer’s instructions, enzyme-linked im-
munosorbent assays (ELISA) were used to measure salivary and
plasma cortisol levels (Cortisol Parameter assay kit, R&D), plas-
ma IL-10 levels (Human IL-10 HS ELISA, Bender MedSystems),
plasma dehydroepiandrosterone (DHEA) levels (DHEA-ELISA,
Biosource) and plasma IL-6 levels (Human IL-6 Quantikine HS
ELISA, R&D).
For the quantification of neuropeptide Y (NPY) levels, peptide
extraction from plasma was conducted based on the manufactur-
er’s instructions using C18 separation columns (Phoenix Pharma-
ceuticals Inc.) followed by lyophilization and freezing. The extract
was then reconstituted and assayed for NPY levels using competi-
tive ELISA (Phoenix Pharmaceutical Inc.). Based on the manufac-
turer’s report, the intra-assay coefficient of variation for all ELISA
kits is 6.9–10%, as was indeed observed in our samples.
Psychological and Cognitive Assessments and Stress
Manipulations
The psychological stress paradigm continued throughout the
sleep-deprived night and included physiological, social and cog-
nitive tasks, which will be described in detail in a separate paper
focusing on the psychological and cognitive outcomes of the
study. Shortly, participants had to complete computerized tasks
while putting their nondominant hand in ice-cold water; were
engaged in a social stress procedure, which was based on the
Leaderless Group Discussion [41] and included elements of com-
petition, cooperation, responsibility and social status, and were
subjected to several cognitive tasks, such as mental arithmetic
Tel-Aviv University Lib. of Life Science and Med.
tasks [42].
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DOI: 10.1159/000348698
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 50
Monday
Friday
45
40
State anxiety
35
30
25
20
a Stress Control
Fig. 2. a Comparison of state anxiety levels between the control and
sleep-deprived groups before (Monday) and after (Friday) the par-
adigm. Significant differences were evident within the sleep-de-
prived group participants, but not within the control group par-
ticipants. Means ± SEM, p < 0.05. b Comparison of depression
A battery of both written and computerized tests was used to
assess trait and state anxiety, depression symptoms, dissociation,
behavior in stress situations, and coping styles and resilience. Ad-
ditionally, public presentations and social stress tasks were con-
ducted during the night hours. All of the tests and tasks were given
in their validated Hebrew versions and included the State-Trait
Anxiety Inventory-Trait Scale (STAI-T), State-Trait Anxiety In-
ventory-State Scale (STAI-S), patient health questionnaire (PHQ-
9), Clinician Administered Dissociative State Scale, analog mood
scales, dot probe, continuous performance test, text presentation
and social stress tasks.
Data from the STAI-T, STAI-S and PHQ-9 were also used for
validating the psychological effect of the stressor, and thus these
tests are described in detail below:
The STAI [43] consists of two 20-item scales measuring situ-
ational variations in levels of perceived anxiety (state anxiety).
Likert scales ranged from 1 (not at all/almost never) to 4 (extreme-
ly/almost always) and scores ranged from 20 to 80, with higher
scores reflecting higher anxiety. The translation of these scales to
Hebrew was done according to the instructions of Spielberger and
Sharma [44] and was found to be reliable and valid for use with
Israelis [45].
The PHQ-9 [46] is a self-reported depression rating scale con-
sisting of 9 items on which the diagnosis of DSM depressive dis-
order is based (designed to assess the severity of depressive
symptoms). Reliability and diagnostic validity of the PHQ-9
were established in several studies [46, 47].
Statistical Analysis
Data from both replicates were combined for the analysis, as
no noticeable differences between the two replicates were ob-
served. For each index examined, a 2 × 2 × 2 repeated-measure
198 Neuroimmunomodulation 2013;20:194–204
DOI: 10.1159/000348698
12
Monday
Friday
10
Depression (PHQ-9)
8
6
4
2
0
b Stress Control
levels, as indicated by PHQ-9 results, between the control and
sleep-deprived groups before (Monday) and after (Friday) the par-
adigm. Significant differences were evident within the sleep-de-
prived group participants, but not within the control group par-
ticipants. Means ± SEM, p < 0.01.
analysis of variance with a predetermined significance level of
0.05 was conducted. The independent between-participant vari-
ables were group (stress/control) and sex (male/female). Time
(before and after stress exposure) served as a repeated within-sub-
ject factor. Specific data observations that deviated from their
group mean by more than 3 SDs were excluded from statistical
analysis. These are identified in the Results section. Provided sig-
nificant group differences were found, Fisher’s protected least sig-
nificant difference analyses were performed to compare specific
pairs of groups based on a priori hypotheses. All statistical analy-
ses were conducted using StatView software (SAS Institute, San
Francisco, Calif., USA).
Results
Stress Manipulation Check
Significant group-by-time interaction effects were
found for state anxiety (F1, 39 = 11.99, p < 0.01; fig. 2a) and
depression (as indicated by PHQ-9 results; F1, 39 = 17.96,
p < 0.01; fig. 2b). Additionally, no differences were found
between the two experimental groups for trait anxiety.
These findings indicate a robust psychological impact of
the experimental procedure, as they indicate an immedi-
ate change in state anxiety and depression within the
stress group only, which was not evident at all in the trait
anxiety data. We further examined these findings using
paired Student’s t tests between the two experimental
Tel-Aviv University Lib. of Life Science and Med.
groups, which indicated no differences before the para-
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Table 1. Results of psychological measures assessed 3 days before the paradigm and on the morning following it

Index Sex Stress Control Statistical

n n outcome
Monday Friday Monday Friday

State anxiety male 9 30.15±2.49 46.82±5.58 7 38.43±3.98 38.71±5.76 *

female 14 31.78±2.77 39.56±2.34 11 33.98±2.43 31.45±3.60
Trait anxiety male 9 41.07±4.72 33.86±4.27 7 38.86±3.37 47.00±5.33 NS
female 14 40.53±2.14 33.56±3.19 11 43.55±2.51 36.91±2.42
PHQ-9 male 9 4.93±1.84 9.79±2.21 7 9.14±1.93 6.14±1.68 *
female 14 4.44±0.82 8.81±1.49 11 6.05±1.04 5.82±1.08

Means ± SEM of the different psychological measures before (Monday) and after (Friday) sleep deprivation, and statistical outcomes
indicating significant differences. * These measures showed significant interaction between group and time point, resulting in elevated
anxiety and PHQ-9 in the stress group following the paradigm. NS = No significant outcome.

Table 2. Results for white blood cell counts assessed 3 days before the paradigm and on the morning following it

Cell population Sex Stress, cells/ml Control, cells/ml Statistical

outcome
n Monday Friday n Monday Friday

Lymphocytes male 8 1,613±120 1,718±151 7 2,023±331 2,075±383 NS

female 14 1,750±150 2,261±251 11 1,918±103 2,199±184
Monocytes male 3 304±67 103±28 4 395±122 137±23 *
female 5 181±15 88±17 3 109±20 52±5
Granulocytes male 8 2,142±306 2,529±259 6a 2,216±350 2,056±361 NS
female 14 2,101±266 1,798±222 11 2,139±147 2,096±283
NK male 8 96±17 74±15 7 117±19 98±20 NS
female 14 82±11 70±11 11 74±9 66±9

Means ± SEM of white blood cell subpopulation count before (Monday) and after (Friday) sleep deprivation, and statistical outcomes
indicating significant differences. * Monocyte counts were significantly higher in males than in females regardless of group and time
point. NS = No significant outcome. a One male deviant subject was removed from the control granulocyte group.

digm and significant differences following it (p < 0.01).
All findings are presented in table 1.
Immunological Indices
Assessments of NK cytotoxicity and FACS analysis
were conducted on all 23 stress and 18 control partici-
pants, excluding 1 male subject from the stress group in
whom the second blood withdrawal was unsuccessful.
For all cytokine and hormone measurements, we ran-
domly and a priori chose 19–22 participants from the
stress group, and matched those to 13–17 participants
from the control group, enforcing similar representation
for each replicate and sex. We conducted this random
selection in order to keep within the available number of
lem with one set of tubes, IL-6 and monocytes were only
assessed in approximately half of the samples.
Leukocyte Counts. The experimental paradigm had no
effect on the total leukocyte numbers or on the number
of any leukocyte subtype. Further examination of cellular
marker expression levels (see Methods) on all related leu-
kocyte subtypes yielded also no effect for sleep depriva-
tion, although baseline levels were within the detection
limits and indicated individual differences. The number
of lymphocytes, monocytes and granulocytes did not sig-
nificantly differ between the groups, nor between baseline
values (Monday) and values after sleep deprivation (Fri-
day) in both groups. When examined for gender differ-
ences, males had a significantly higher number of mono-
Tel-Aviv University Lib. of Life Science and Med.

wells in the ELISA plates. Also, due to a technical prob- cytes than females, independently of group condition
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Stress and Sleep Deprivation DOI: 10.1159/000348698
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 80

70

60

K562 cytotoxicity (%)

50

40

30

20
Control Fri.
Stress Mon.
10 Control Mon.
Stress Fri.
0
E:T 1 E:T 2 E:T 3 E:T 4 E:T 5 E:T 6 E:T 7 E:T 8
a E:T ratio (whole blood:K562)

80

70

60
K562 cytotoxicity (%)

50

Fig. 3. a Comparison of NK cytotoxicity 40

against K562 cells between the sleep-de-
prived and the control group before (Mon- 30
day) and after (Friday) the paradigm. No
significant differences were evident be- 20
tween groups or time points. Means ± SEM. Male Fri.
Male Mon.
b Comparison of NK cytotoxicity against 10 Female Fri.
K562 cells between males and females be- Female Mon.
fore (Monday) and after (Friday) the sleep 0
deprivation paradigm. Significant differ- E:T 1 E:T 2 E:T 3 E:T 4 E:T 5 E:T 6 E:T 7 E:T 8
ences were evident between males and fe- b E:T ratio (whole blood:K562)
males, but not between the two different
time points. Means ± SEM, p < 0.01.

(F1, 11 = 10.89, p < 0.01). No additional gender differences
were found among the other leukocyte subpopulations
examined. All data are presented in table 2.
NK Cell Counts and Cytotoxic Activity. The experi-
mental paradigm neither affected NK numbers or cyto-
toxicity between the groups nor between both time points
(at baseline and after sleep deprivation). When examined
for gender differences, males had significantly higher kill-
ing activity levels than females, independent of group
condition (F1, 36 = 8.49, p < 0.01; fig. 3).
Hormone Levels. The experimental paradigm had no
blood NPY levels between the groups, nor between levels
at baseline and after sleep deprivation. Additionally, no
gender differences were found among these hormone lev-
els. All levels are presented in table 3, including minimal
detection levels.
Cytokines. The experimental paradigm had neither an
effect on IL-6 and IL-10 levels between the groups, nor
between levels at baseline and after sleep deprivation. No
significant sex differences were found in these indices. All
levels are presented in table 4, including minimal detec-
tion levels.
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effect on DHEA levels, blood and saliva cortisol levels and

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Table 3. Results of hormonal levels (in ng/ml) assessed 3 days before the paradigm and on the morning following it

Hormone Minimal Sex Stress Control Statistical

detection level n Monday Friday n Monday Friday outcome

DHEA 0.37 male 8 5.34±0.92 7.39±3.42 7 5.28±1.13 4.37±0.81 NS

female 14 2.85±0.31 4.19±0.79 9 2.81±0.47 4.33±1.09
Plasma cortisol 0.156 male 7 13.74±2.03 14.06±2.04 5 10.87±2.69 12.59±1.96 NS
female 14 13.75±2.29 12.69±1.66 8 10.61±3.54 17.77±2.56
Saliva cortisol 0.156 male 8 1.14±0.61 0.72±0.12 7 0.58±0.07 1.29±0.46 NS
female 14 0.60±0.09 0.53±0.07 10 0.92±0.19 0.92±0.21
NPY 0.01 male 8 0.26±0.14 0.25±0.06 7 0.24±0.08 0.21±0.05 NS
female 14 0.34±0.10 0.26±0.04 10 0.20±0.06 0.31±0.03

Means ± SEM of hormonal levels before (Monday) and after (Friday) sleep deprivation, and statistical outcomes indicating signifi-
cant differences. NS = No significant outcome.

Table 4. Results of cytokine levels (in pg/ml) assessed 3 days before the paradigm and on the morning following it

Cytokine Minimal Sex Stress Control Statistical

detection level n Monday Friday n Monday Friday outcome

IL-6 0.156 male 4 0.86±0.24 0.38±0.19 4 1.19±0.86 0.64±0.53 NS

female 5 0.82±0.28 0.71±0.39 4 2.15±0.75 1.78±1.18
IL-10a 0.39 male 7 1.00±0.67 0.76±0.53 5 0.17±0.08 0.12±0.04 NS
female 12 0.21±0.04 0.15±0.04 8 0.15±0.04 0.14±0.08

Means ± SEM of cytokine levels before (Monday) and after (Friday) sleep deprivation and statistical outcomes indicating significant
differences. NS = No significant outcome.a Most, but not all, readings of IL-10 levels were below the minimal detection level provided
by the manufacturer.

Discussion pected physiological levels and individual differences.

The combination of 30-hour sleep deprivation and so-
cial stress tasks was shown to be an effective psychologi-
cal manipulation, as reflected by elevated levels of state
anxiety and depression in the sleep deprivation group on
the morning following the paradigm. However, this po-
tent manipulation had no detectable influence on any of
the neuroendocrine or immunological indices studied
on the morning following the paradigm. Specifically, no
effects were evident regarding (i) levels of NK activity;
(ii) numbers of leukocyte subsets (including monocytes,
granulocytes, lymphocytes and NK cells); (iii) expression
levels of various activation markers (including NKG2D,
MHC-II, CD11a and FasL) on leukocytes subsets, and
(iv) cytokine and hormone levels (IL-6, IL-10, NPY,
DHEA, and saliva and plasma cortisol). Importantly, the
great majority of the indices tested were within the detec-
For example, the NK cytotoxicity assay yielded signifi-
cant killing, increasing specific lysis from 6 to 70% along
the different E:T ratios, and hormone levels are within
normal and detectable levels (as detailed in table 3).
To further validate the methodological integrity of the
data, we analyzed our findings for sex differences, as some
were previously reported. Specifically, and as was previ-
ously shown by us and others [27, 48], we found a sig-
nificantly higher NK cytotoxic activity in males than in
females. Additionally, higher numbers of monocytes
were observed in males than in females, as was also re-
ported by others [49, 50]. Importantly, these differences
did not interact with sleep deprivation or with the time
points tested (before and after the experimental para-
digm). Overall, these findings suggest resilience of the im-
mune system of young students to our sleep deprivation/
stress paradigm. This apparent resilience concurs with
Tel-Aviv University Lib. of Life Science and Med.

tion levels of the different bioassays and showed the ex- the ability of young people to cope with occasional sleep
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Immune Resilience in Students following Neuroimmunomodulation 2013;20:194–204 201

Stress and Sleep Deprivation DOI: 10.1159/000348698
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deprivation and temporary stressful events, maintaining
their health and resistance to opportunistic diseases, as
would be evolutionarily sound in young adults.
When comparing our study design to common sleep
deprivation studies, in which significant immunological
impacts were reported [12, 51, 52], one can notice four
major differences that may account for the apparent in-
congruence in results. In our study we: (i) measured in
vivo plasma cytokine levels, as opposed to assessing in vi-
tro ‘induced production’ levels; (ii) allowed participants to
maintain normal daily routine following baseline assess-
ment and up until the beginning of the sleep deprivation
period, rather than restricting them to the laboratory; (iii)
added social and physiological activities during the sleep
deprivation period, as opposed to keeping participants
under conditions of social isolation and minimal physical
activity during the sleep deprivation period, and (iv) as-
sessed immune indices following completion of the entire
paradigm, when participants were ready to resume their
daily activity rather than assessing immunity at specific
time points along the night period and in close conjunc-
tion with the potentially acute stress responses.
In the current study, we directly measured plasma lev-
els of cytokines and hormones. This approach is different
from most of the studies addressing cytokine levels under
stress [10, 53], which use the in vitro ‘induced production’
approach that is based on in vitro activation of white blood
cells (or of a specific subpopulation) employing substanc-
es that mimic bacterial or viral infections (such as lipo-
polysaccharides or CpG-C). This in vitro activation leads
to the secretion of cytokines from target cells, hence its
advantage, but it is also biased since it reflects the specific
leukocyte composition of the sample at the exact time of
blood withdrawal and its potential to secrete specific cyto-
kines upon activation. It does not indicate plasma levels of
the measured cytokines, which are derived from many
sources along an extended period. It is noteworthy that
plasma baseline levels of most cytokines are commonly
very low or below detection levels, and thus the ‘induced
production’ approach is commonly used. Importantly,
studies by others and us have shown that plasma cytokine
levels may show different patterns of effects (even oppo-
site) from those observed following ‘induced production’
in the context of some manipulations [54, 55].
It can also be proposed that the current paradigm of
sleep deprivation combined with psychological, cogni-
tive and physiological stress might have only provoked a
priming effect on the immune system [56]. Such priming
effects may only be revealed by approaches such as in
vitro cytokine ‘induced production’, but not by direct
202 Neuroimmunomodulation 2013;20:194–204
DOI: 10.1159/000348698
measurement of plasma levels that was used herein. It is
possible that higher levels of sleep deprivation and stress
exposure may lead to an actual state of immune pertur-
bation, which is reflected by altered plasma levels of cy-
tokine and hormones, and by changes in NK activity and
other immune functions.
Most sleep deprivation studies are conducted in the
setting of a well-controlled sleep laboratory, in which par-
ticipants are continuously present from the beginning of
the study to its end [10, 12, 57]. In the current study, how-
ever, participants were allowed to maintain their normal
daily routine from the day of baseline measurements
(Monday) to the beginning of the sleep-deprived night
(Thursday). Depriving participants from their daily rou-
tine combined with the exposure to the stringent labora-
tory setting during the sleep deprivation period may have
substantial psychological impacts. These psychological
factors may interact with or underlie some of the report-
ed effects of sleep deprivation, effects that were absent in
the current study.
Another major difference characterizing our study was
the social setting along the sleep-deprived night. In most
studies, participants are isolated in the sleep laboratory,
with some studies even maintaining minimal movement
policy [12, 53]. In the current study, participants spent
their sleep-deprived night together, interacting socially,
and were allowed to move freely in the laboratory area. As
social isolation, even for short periods of time, was report-
ed to decrease immune activity [58, 59], it is possible that
the experience of social isolation, which characterizes
most sleep deprivation studies, modulated immunologi-
cal outcomes, leading to higher inflammation markers
and lower NK cytotoxic activity [60]. Inversely, social in-
teractions in our study may have buffered or counteracted
the impacts of sleep deprivation and stress.
Additionally, some researchers suggested that in con-
trast to the known immunosuppressive effects of chronic
stress, short-term (acute) stress actually leads to immu-
noenhancement [61]. For example, it was shown that a
short paradigm of acute stress in mice prior to UVB ra-
diation exposure improved anti-tumor activity com-
pared to control conditions [25]. As our paradigm in-
cluded exposure to several acute stressors during the
sleep-deprived night, it is possible that these tasks in-
duced immunoenhancing effects, which in turn counter-
acted immunosuppressive effects of sleep deprivation.
Last, it is also possible that the herein lack of sleep de-
privation-/stress-related immunological changes results
from the specific time points that were chosen for blood
Tel-Aviv University Lib. of Life Science and Med.
collection. Unlike other studies, in which blood samples
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were collected during the night period [62], in our study
blood and saliva were collected in the morning hours fol-
lowing the sleep-deprived night. The blood and saliva col-
lection time point was chosen in this study to reflect the
overall cumulative immunological and psychological ef-
fect of the paradigm, in contrast to assessing acute and
potentially transient perturbations. Thus, it is possible
that in the current study sleep deprivation had also led
to immunological perturbations, but these were short
lasting and no longer present at the time of sample collec-
tion. Specifically, the leukocyte sample composition,
which markedly determines in vitro cytokine induced
production levels and NK activity, constantly fluctuates
based on momentary changes in stress hormone levels
[63]. Engaging in normal social activity within the labora-
tory before the second blood sampling may have masked
these previous temporary perturbations, reflecting only
long-lasting effects with potential biological significance.
Clearly, this study has several shortcomings, especially
given its lack of significant results. Immune indices were
not assessed during the night period, as is common in
most studies. The rationale for this approach was our aim
to study the cumulative overall effects of the sleep-de-
prived night along with stressful tasks evident on the
morning following it rather than studying periodical
acute alterations during the night. Cytokine levels were
only measured in the plasma, as we believe this approach
better reflects in vivo levels and their biological ramifica-
tions. Also, the study was limited to young healthy stu-
dents, without comparing them to more susceptible pop-
ulations. Last, only one level of sleep deprivation and
stress exposure was used, and the two manipulations were
imposed simultaneously and not studied separately.
However, the findings are sufficient to suggest a resil-
ience of the immune system in young students to a com-
bination of sleep deprivation and a stressful situation.
The evident resilience is demonstrated herein when test-
ing immunity following the completion of the experi-
mental paradigm at an active daytime period. Although
fluctuations in immune indices could have occurred dur-
ing the night period, they do not seem to have long-last-
ing effects in the current study and may thus bear mini-
mal biological significance. That said, studies have also
reported greater susceptibility to stress in older adults [64,
65], and in such populations both the ongoing fluctua-
tions and the long-term effects on immune measures
should be further studied.

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