behavioral
sciences
ArticleSex Differences in Salivary Oxytocin and Cortisol
Concentration Changes during Cooking in a
Small Group
Teruko Yuhi 1, * , Kosuke Ise 1 , Kei Iwashina 1 , Naoya Terao 1 , Satoshi Yoshioka 1 ,Keijiro Shomura 1 , Toshikatsu Maehara 1 , Akari Yazaki 1 , Kana Koichi 1 , Kazumi Furuhara 1 ,Stanislav M. Cherepanov 1 , Maria Gerasimenko 1 , Anna A. Shabalova 1 , Kouhei Hosoki 1 ,Hikari Kodama 1 , Hong Zhu 1 , Chiharu Tsuji 1 , Shigeru Yokoyama 1 and Haruhiro Higashida 1,2
1

2*

Department of Basic Research on Social Recognition, Research Center for Child Mental Development,
Kanazawa University, 13-1 Takara-Machi, Kanazawa 920-8640, Japan; kou.conan@gmail.com (K.I.);
kei.i@stu.kanazawa-u.ac.jp (K.I.); teraomedical058@gmail.com (N.T.); apollokun23@gmail.com (S.Y.);
shomuracheckmate@gmail.com (K.S.); dmugen624@gmail.com (T.M.); yazakitty1995@gmail.com (A.Y.);
cartah3beans@gmail.com (K.K.); furururukz.999@gmail.com (K.F.); stas4476@mail.ru (S.M.C.);
mgera_08@mail.ru (M.G.); ashabalova@me.com (A.A.S.); khosoki473@gmail.com (K.H.);
hikari1223.xas@gmail.com (H.K.); julia5444482@hotmail.com (H.Z.); ctsuji@med.kanazawa-u.ac.jp (C.T.);
shigeruy@med.kanazawa-u.ac.jp (S.Y.); haruhiro@med.kanazawa-u.ac.jp (H.H.)
Laboratory for Social Brain Studies, Research Institute of Molecular Medicine and Pathobiochemistry, andDepartment of Biochemistry, Krasnoyarsk State Medical University named after Prof. V. F. Voino-Yasenetsky,
660022 Krasnoyarsk, Russia
Correspondence: y-teruko@med.kanazawa-u.ac.jp; Tel.: +81-076-265-2457; Fax: +81-076-234-4213

Received: 1 October 2018; Accepted: 23 October 2018; Published: 3 November 2018


Abstract: Background: Oxytocin (OT), a neuropeptide, has positive effects on social and emotional
processes during group activities. Because cooking is an integrated process in the cognitive, physical,
and socio-emotional areas, cooking in a group is reported to improve emotion and cognition.
However, evidence for efficacy in group cooking has not been well established at the biological level.
Methods: To address this shortcoming, we first measured salivary levels of OT and cortisol (CORT),
a biomarker of psychological stress, before and after group cooking for approximately 1 h by people
who know each other in healthy married or unmarried men and women. We then compared theinitial OT and CORT concentrations with those during individual non-cooking activities in isolation.
Results: Baseline OT concentrations before group and non-group sessions did not significantly
differ and OT levels increased after both types of activity in men and women. In men, however,the percentage changes of OT levels in the first over the second saliva samples were significantly
small during cooking compared with those in individual activities. In women, however, such a
difference was not observed. In contrast, the mean salivary CORT concentrations after group cooking
were significantly decreased from the baseline level in both sexes, though such decreases were
not significant after individual activity sessions. The sex-specific differences were marital-statusindependent. Conclusion: These results indicate that OT and CORT concentrations after two activity
sessions by a familiar group changed in opposite directions in a sex-specific manner. This suggests
that, because cooking is experience-based, we need to consider the sex-specific features of groupcooking if we apply it for intervention.
Keywords: oxytocin; cortisol; group activity; in-group member; cooking; stressBehav. Sci. 2018, 8, 101; doi:10.3390/bs8110101

www.mdpi.com/journal/behavsci

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1. Introduction
The nonapeptide oxytocin (OT) promotes social affiliation and bond formation, and functions to
reduce anxiety and dampen the stress response in mammalian species [1–15]. OT also plays a role inmediating the stress-buffering and anxiolytic effects of close social interactions [16,17]. Impairmentsin the OT system are found in patients with autism spectrum disorder (ASD), borderline personality
disorder, and anxiety disorders [3,18–23]. Nasal application of OT in subjects with ASD with or without
comorbid intellectual disability has been shown to improve social interactions [24–31].
One way to understand the OT system in the nervous system is to measure OT concentrationsin the brain or cerebrospinal fluid (CSF). We have reported that OT is released into the brain duringsocial stress in mice [32]. Furthermore, OT concentrations in the CSF or extracellular fluid correlated
positively with OT concentrations in blood plasma after experimentally induced stress, though baseline
concentrations in blood may be reflected less in those in CSF [33]. As invasive methods are hard
to apply in living humans, an alternative more practical non-invasive method is to measure OT
concentrations in blood or salivary samples [34–44].
Pleasant activities in a group have beneficial effects in humans [45]. Therefore, not only during
stressful conditions, it is hypothesized that OT can be released during pleasant activities in groups,
especially with familiar members. There are several interesting reports on singing in a group [46–49].
Contrary to the initial expectation from the reports on the effects of singing referenced above, salivary
OT increases during solo singing but not in singing as part of a choir [46]. This suggests that, althoughgroup singing seems to be an enjoyable experience and one that facilitates bonding [49], it can also berather stressful because of having to pay attention to and account for the voices of other singers. In
another case, we recently examined the biological effects of Japanese taiko drumming in a group as partof an educational intervention for children who are emotionally disturbed because of maltreatment.
Taiko drumming can apparently improve the social behavior of these children, as judged by caregivers
and school teachers [41]. Our results showed that the children’s mean salivary OT concentrations
increased to various degrees after the recital but not much rehearsals. Group musical instrumental
playing and choir singing are also reported to improve cognitive function, communication, impaired
behavior, and mood [40,41,50–58], although the effects are not always consistent [59]. Therefore,
we need to re-ask the question how group activity is simply comfortable to people by monitoringbiomarkers, from the view of stress produced by group activity.
Cooking is an essential activity for daily living and a familiar activity for most people. Home
cooking tasks are performed by one individual or by family members for the benefit of the family.
Cooking is an integrated process in the cognitive, physical, and socio-emotional areas; therefore, kitchenskills or behaviors are useful for diagnosing cognitive function in geriatric psychiatry patients [60,61].
Furthermore, cooking is used as an intervention tool in therapy and rehabilitation [62–64]. Several
studies that conducted cooking activities using people with dementia report statistically significant
improvements in cognition and depression [63,65], because cooking is experience-based and pleasantfeelings, while cooking may be ambivalent, i.e., pleasant and/or stressful.
To our knowledge, the beneficial effects of group cooking have not been measured by monitoring
OT concentrations. Therefore, we first arranged a group cooking activity in a very small group as a pilot
study. The group consisted of male and female healthy volunteers who were students and laboratory
workers at a medical university who got acquainted in the authors’ laboratory. The activities werecarried out during educational sessions as part of the curriculum during a 3-year period (2015–2017).
After we noticed that, in men, increases in OT concentrations after cooking were not high in trials
during 2015 and 2016, we added one experimental item in 2017 to measure saliva cortisol (CORT)
concentrations, a biomarker of psychological stress, in the same saliva samples [66]. This was done
to understand to what extent cooking is differentially stressful between men and women. In general,cooking is likely to be a pleasant activity for women but not for men. In addition, because cooking
skill may not be similar between married and single people, we analyzed changes in OT and CORT
concentrations by classifying participants by marital status in both sexes. Finally, we measured theseBehav. Sci. 2018, 8, 101

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concentrations during individual (free, usually desk or bench work) activities in isolated conditions
for comparison.
2. Materials and Methods
2.1. Participants
The study recruited 31 healthy volunteer participants with an age cut-off of 21–69 years. Of the 31
recruited, we selected 18 (9 men [27.6 ± 5.3 years old] and 9 women [33.9 ± 3.9 years old], Table 1), forfurther statistical analysis based on more than 10 measures of OT and 5 measures of CORT (participants
were third-year undergraduate students at Kanazawa University School of Medical Sciences andgraduate students from Osaka University United Graduate School for Mental Development at the
Kanazawa Campus. Others were workers and teachers from the Kanazawa University Research Centerfor Child Mental Development who engaged in educational sessions.) Ethnically, 14 participants
were Japanese, 3 were Russian, and 1 was Chinese. Six participants were married and 12 were single(Table 1). During the tests, all participants got acquainted in the one author’s laboratory and became
friends while staying together for 8 weeks to 12 years, as language or culture barriers decreased.
Table 1. Demographic data.
Men (Married)

Women (Married)

Number of participants

9 (2)

9 (4)

Each year
2015
2016
2017

3 (1)
4 (2)
5 (2)

4 (3)
6 (4)
9 (4)

Age (years ± s.e.m. (range)) a27.6 ± 5.3 (21–69)

33.9 ± 3.9 (21–50)

Education (years ± s.e.m. (range)) b

16.4 ± 0.96 (15–22)

16.1 ± 0.54 (14–20)

8 (1)
1 (1)
0

6 (3)
2 (0)
1 (1)

Ethnicity
Japanese
RussianChinesea p = 0.3466, two-tailed Student’s t-test; b p = 0.7657, two-tailed Student’s t-test.

2.2. Ethics Statement
The study was approved by the student curriculum committee of Kanazawa University Graduate
School of Medical Sciences in 2014 as educational sessions. The study was carried out during November
to December in 2015, 2016 and 2017. All procedures involving human participants were conductedin accordance with the ethical standards of the institutional research committee and with the 1964
Helsinki Declaration. The participants were told that they could choose not to supply their saliva oneach occasion.
2.3. Assessment
The participant’s salivary OT or CORT levels were assessed during 27 group cooking tasks and
11 sessions of individual activities other than cooking, such as desk or bench work (Supplementary
Figure S1). This was done over 3 years, approximately twice a week. Experiments were started around
13:00. Saliva was collected during the first 10 min. At 2 to 5 min after participants rinsed their mouths
with water, when their mouths were filled with newly secreted saliva, they bit down on a sterile 15-mL
polypropylene tube (Greiner Bio-one Co. Ltd., Tokyo, Japan) and secreted saliva directly into the tube
by chewing for 2–4 min [40,41]. This method was less stressful than using a cotton swab. Then they
participated in an approximately 60-min cooking session in one room or in a 60-min individual activity

Behav. Sci. 2018, 8, 101

4 of 15in places of their choosing. Each cooking session was held with 4–10 participants (Supplementary
Figure S1). Saliva was collected a second time 10 min after the end of each session.
2.4. Saliva Collection and Analysis
The saliva samples (approximately 0.5–1.0 mL) in polypropylene tubes were kept on ice. They
were then immediately frozen and stored at −80◦ C, as described previously [41]. At the assay time,they were thawed and centrifuged twice at 4 ◦ C at 1500× g for 15 min. The samples were divided into
1.5-mL microtubes, each containing 100 µL [67].
Salivary OT was measured using a 96-plate commercial OT-ELISA kit (Enzo Life Sciences,
Farmingdale, NY, USA), as described previously [41]. Saliva was not extracted in the current
experiment because some studies have reported conducting measurements without extraction [40,68],
as validated by MacLean et al. [69,70].
Measurements were performed in duplicate. The optical density of the samples and standards wasmeasured at wavelengths of 405 and 590 nm by a microplate reader (Bio-Rad, Richmond, CA, USA).
Sample concentrations were calculated by MatLab-7 according to the relevant standard curve [40].
It has been reported that some saliva components interact with the labelled OT in the assay
mixture [69,71]. To test this, we first measured samples spiked with OT (0–250 pg/mL). A
very high level was observed with no addition of spiked OT, suggesting an interaction with
nonspecific antibody-interacting substances. Even with interacting substances in the samples, theenzyme immunoassay monitored concentrations were proportional to the spiked OT concentrations(Supplementary Figure S2), suggesting that monitored values are useful for calculating the ratio
between values. Therefore, the difference between OT concentrations before and after the sessions
seems to be more reliable.
2.5. Salivary cortisol assay
The same samples used for OT measurement were also used to measure CORT. Determinationof saliva CORT was performed using a cortisol enzyme immunoassay kit (Salimetrics, State College,
PA, USA), as described by Kumazaki et al. [67]. Samples (25 µL) were treated according to the
manufacturer’s instructions. Measurements were performed in duplicate. The optical density of thesamples and standards was measured at a wavelength of 450 nm by a microplate reader (Bio-Rad).
Sample concentrations were calculated by MatLab-7 according to the relevant standard curve [40].
2.6. Statistical Analysis
Two-tailed Student’s t-tests were used for single comparisons between two groups. One- or
two-way analyses of variance (ANOVA) were used for data with two or three components, respectively.
Post hoc comparisons were performed only when the main effect was statistically significant. The
p-values of the multiple comparisons were adjusted using Bonferroni’s correction. All data fromin vivo and in vitro studies are shown as means ± s.e.m. In all analyses, p < 0.05 was taken to indicate
statistical significance. All the analyses were performed using STATA data analysis and statisticalsoftware (Stata Corp. LP, College Station, TX, USA).
3. Results
3.1. Salivary Oxytocin Concentrations
Table 1 shows the characteristics of the participants in the group cooking study. Nine men(eight Japanese and one Russian) and nine women (six Japanese, two Russians and one Chinese)
who were university students, postgraduate students, laboratory technicians and professors from two
universities took part in the study in each November and December from 2015 to 2017.
The OT concentrations in the saliva collected before cooking in a group and the individual
activity in isolation were determined and used as the baseline OT levels (Table 2). There were noBehav. Sci. 2018, 8, 101

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significant differences among these values (one-way ANOVA, F3,231 = 1.93, p = 0.1250). The salivary
OT concentrations measured before (first salivary samples) and after individual and cooking sessions(second salivary samples) were plotted in the male and female groups (Figure 1A–D). There weresignificant differences observed in the average OT concentrations among the four assessments ofboth sexes (one-way ANOVA, F7,462 = 3.15, p = 0.0029). Bonferroni post hoc tests revealed significant
differences between the OT level after the individual activity in women (185 ± 31 pg/mL, n = 44) andthe OT concentrations before and after cooking in men (121 ± 12, n = 55, 120 ± 14, n = 55, respectively;
p < 0.016 for each value).
Table 2. Baseline concentrations of oxytocin and cortisol.
Men

Women

OT concentrations (pg/mL ± s.e.m.)Individual activities

beforeafter

170 ± 18 (46) a,b
207 ± 24 (46) b,c

185 ± 31 (44) a,b
234 ± 35 (44) b,c

Cooking

beforeafter

125 ± 13 (55) a,b,c
120 ± 14 (55) b,c

153 ± 12 (90) a,b
178 ± 16 (90) b

Individual activities

beforeafter

420 ± 69 (17) d,e,f
278 ± 41 (17) e

286 ± 31 (31) d,e
235 ± 24 (31) e,f

Cooking

beforeafter

330 ± 34 (26) d,e,g
190 ± 18 (26) e,f,g

312± 49 (35) d,e,h
204 ± 21 (35) e,f,h

CORT concentrations (ng/dL ± s.e.m.)a p = 0.1250, one-way ANOVA, among values labelled with a ; b p = 0.0029, one-way ANOVA, among values labelledwith b ; c p < 0.01, Bonferroni post hoc test, for values labelled with c ; d p = 0.1564, one-way ANOVA, among values
labelled with d ; e p = 0.0001, one-way ANOVA, among values labelled with e ; f p < 0.01, Bonferroni post hoc test,for values labelled with f ; g p = 0.0011, two-tailed Student’s t-test, between values labelled with g ; h p = 0.0049,
two-tailed Student’s t-test, between values labelled with h .

The ratio of the salivary OT concentration changes was calculated by the values in the first (before)
saliva over the second (after) saliva samples, as shown in Figure 1. Figure 2A shows percentagechanges in 46 individual and 55 cooking sessions in men. OT concentration changes after cooking weresignificantly lower (two-tailed Student’s t-test, p = 0.0216) than those in the individual sessions. We
further analyzed the average changes of the individual male participants. Figure 2B indicates that OT
concentration increases after cooking were significantly lower (two-tailed Student’s t-test, p = 0.0182,
n = 9 for each session).
The same analysis was performed for OT concentrations obtained in women. There were no
significant differences in OT concentration changes between individual activity and cooking sessions
as plotted in every measurement (Figure 2C, p = 0.1724, n = 43 and 90, respectively) nor in individual
women (Figure 2D; n = 9, p = 0.2204).

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Figure 1. Oxytocin (OT) concentrations before and after a single group (cooking) or non-group(individual activity) session. OT levels are for the first (before) and second (after) salivary samples
collected
fromnine men(OT)
and nine
women engaged
cookinga groupor in(cooking)individual
orFigure1. Oxytocin
concentrations
before inandafter ainsingle
group
or desk
non-group
bench
work
for
approximately
1h.
p-valuesarefor
two-tailed
Student’s
t-tests:(A)
individual
activity(individual activity) session. OT levels are for the first (before) and second (after) salivary samples
by men,p = 0.2200,
n = 46;(B)andcookingmen, engaged
p = 0.8079,= 55; (C)inindividual
by women,collected
from ninemennine by
women
in ncooking
a group oractivity
in individualdesk orp = 0.2935,
n
=
44;
(D)
cookingbywomen,
p=
0.2086,n=90.
bench work for approximately 1 h. p-values are for two-tailed Student’s t-tests: (A) individual

activity by men, p = 0.2200, n = 46; (B) cooking by men, p = 0.8079, n = 55; (C) individual activity by

Figure 2 shows marital status by indicating single (open symbols) or married (filled symbols)
women, p = 0.2935, n = 44; (D) cooking by women, p = 0.2086, n = 90.
individuals. Judging from the bar graphs, the two symbols seem to be unevenly distributed and do notform any subpopulations. There were no differences between the average values in OT concentration
Figure 2 shows marital status by indicating single (open symbols) or married (filled symbols)changes before and after each activity in the married and single groups (Table 3). Two-way ANOVA
individuals. Judging from the bar graphs, the two symbols seem to be unevenly distributed and do
showed no significant interaction between marital status and sessions on OT (F3,227 = 0.42, p = 0.7937).
not form any subpopulations. There were no differences between the average values in OT
The results clearly indicate that OT concentration changes before and after cooking is correlated withconcentration changes before and after each activity in the married and single groups (Table 3).
male participants, but not to marital status. Therefore, in the last year (2017), we measured CORT
Two-way ANOVA showed no significant interaction between marital status and sessions on OT
levels in the saliva to test sex-relatedness in stress during cooking.
(F3,227 = 0.42, p = 0.7937). The results clearly indicate that OT concentration changes before and after
cooking is correlated with male participants, but not to marital status. Therefore, in the last year(2017), we measured CORT levels in the saliva to test sex-relatedness in stress during cooking.

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Figure2. Percentagechangesin oxytocinlevelslevels
before before
and aftera singleof oxytocinof
Figure2. Percentagechangesin oxytocin
andafter session.
a singlePercentage
session. Percentage
levelsfor thefirst for(before)salivaover thesecondsaliva(after)
samples
aftersamples
each session.
Salivawas
oxytocinlevelsthe first(before)salivaover(after)
the second
salivaafter eachsession.
collected
duringindividual(non-cooking)
and
group
(cooking)
activities
in
men
(A)
and
women
Saliva was collected during individual (non-cooking) and group (cooking) activities in men (A)(C).
andThe
averageof oxytocinlevelsfor the first(before)the saliva
secondover(after)women(C).percentage
The average
percentageof oxytocinlevelsfor thesaliva
first over
(before)thesaliva
secondsamples
individual
male(B) and female
are for two-tailed
t-tests:(after) insalivasamples
in individual
male(D)(B)participants.
and female p-values(D) participants.
p-values Student’s
are for two-tailed(A)p=0.0216
betweenindividual
activity
(n
=
44)
and
cooking
(n
=
55)
in
men;
(B)
p
=
0.0492,
betweenStudent’s t-tests: (A) p = 0.0216 between individual activity (n = 44) and cooking (n = 55) in men; (B) p
individual(n individual= 9) and cooking(n =(n9)=in9)men;
p = 0.1849
individual= 44)= 0.0492,activity
betweenactivity
and(C)
cooking(n =between9) in men;(C) p =activity
0.1849 (nbetweenandcooking(n=
90)
inwomen;(D)p=
0.6373
betweenindividual
activity
(n
=
9)
and
cooking(n= 9)individual activity (n = 44) and cooking (n = 90) in women; (D) p = 0.6373 between individual activityin(nwomen.
Filledandopensymbols
represent
marriedandsingleparticipants,
respectively.
= 9) and cooking (n = 9) in women. Filled and open symbols represent married and single

participants, respectively.
Table 3. Percentage of OT and CORT levels for the first (before) saliva over the second (after) saliva
Table 3.
Percentage
of OT
CORT
levelsforparticipants.
the first (before) saliva over the second (after) saliva
samples
after
each session
in andmarriedandsinglesamples after each session in married and single participants.
Men
Women

Men
OT level changed (%±s.e.m.) ab)
148 ± 22 (22)
Individual activities c (Married
Individual activities c (Married b) 148 ± 22 (22)128 ± 12 (24)
Individual activities c (Single b )Individual activities c (Single b)
128 ± 12 (24)b)
102± 12 (22)
Cooking c (Marriedcb
Cooking (Married )102± 12 (22)108 ± 8 (38)
Cooking c (Single b )Cooking c (Single b)
108 ± 8 (38)
CORT level changed (%±s.e.m.) dd
CORT level changed (%±s.e.m.)c (Marriedb)135
42 (7)c (Marriedb) ± 135
Individual
activitiesactivities
Individual± 42 (7)c (Single b )
69
±
14
(10)
Individual
activitiescbIndividual activities (Single )69 ± 14 (10)c (Marriedb)c (Marriedb)
87 ± 12
Cooking
Cooking87(9)± 12 (9)c (Single b )63±
15
Cooking
Cooking c (Single b)
63(17)± 15 (17)
OT level changed (%±s.e.m.) a

Women
153 ± 20 (23)153 ± 20 (23)205 ± 45 (21)205 ± 45 (21)137 ± 12 (55)137 ± 12 (55)152 ± 32 (35)152 ± 32 (35)82 ±
±1010 (14)(14)82
103 ± 9 (17)103 ± 9 (17)60 ±
±88 (15)
(15)
60
82
±
6
(20)
82 ± 6 (20)a A two-way ANOVA shows main effects of marital status of life style (b ; F
p = =0.0396)
two typesa A two-way ANOVA shows main effects of marital status of life 3,235
b; F3,235
style= (2.82,
2.82, pand
= 0.0396)
and(individual and cooking) of sessions (c ; F1,235 = 4.29, p = 0.0396) without a significant interaction between maritalc; F1,235 = 4.29, p = 0.0396) without a significant
two
types
(individual
andcooking)ofsessions(
status and sessions (F3,235 = 0.42, p = 0.7937) on percentage changes of OT concentrations before and after thed A two-way
sessions;
ANOVA
shows
main
effectsof maritalof life(b ; Fon= 3.76, p = 0.0131)
andof
two
interaction
betweenmaritalstatusandsessions(F3,235status= 0.42,p =style
0.7937)changesOT
3,110percentage
c; F
types
of
sessions
(
=
5.13,
p
=
0.0256)
withoutasignificant
interaction
betweenmaritalstatusandsessionsd1,110
concentrations before and after the sessions; A two-way ANOVA shows main effects of marital(F3,110 = 0.67, p = 0.5693) on percentage changes of CORT concentrations before and after the sessions.

status of life style (b; F3,110 = 3.76, p = 0.0131) and two types of sessions (c; F1,110 = 5.13, p = 0.0256)

percentage changes of CORT concentrations before and after the sessions.

3.2. Salivary Cortisol Concentrations
Salivary CORT levels before and after cooking and non-cooking activities in male and female
subjects
and are listed in Table 2. The baseline CORT concentrations before tasks
Behav. Sci. were2018, 8,measured101
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were not significantly different in men and women (one-way ANOVA, F3,105 = 1.79, p = 0.1535).
Furthermore, average CORT concentrations before and after individual activity sessions were not
3.2. Salivary Cortisol
significantly
changedConcentrations
in both men and women: Figure 3A,C; two-tailed Student’s t-test, p = 0.087 (n =17) andp=
0.2074(n =31),
respectively.
However,
percentage changes
cooking
were
Salivary CORT levels
beforeand after cooking
and non-cooking
activitiesafterin maleand female
significantly
in male= 0.0011
female(p = 0.0049(n = 35)) participants,
subjects werelower
measuredand(parelisted (nin =26))
Tableand2. Thebaseline
CORT concentrations
before comparedtasks were
with
prior
basal
values(Figure3B,D).
not significantly different in men and women (one-way ANOVA, F3,105 = 1.79, p = 0.1535). Furthermore,
The CORT
percentageof the salivary
CORTchangeswassessions
calculatedby thein theaverageconcentrations
beforeandconcentration
after individual
activitywere
not values
significantly
first(before)salivaoversecondFigure(after) 3A,C;
salivatwo-tailed
samples, as
shown t-test,in FigureTherenochangedin bothmen
andthewomen:
Student’s
p = 4.
0.087(n =were17) andsignificant
differences
in
CORT
concentration
changesinmenduringboth
individual
andcooking
p = 0.2074 (n = 31), respectively. However, percentage changes after cooking were significantly lower
sessions
as=plotted
in =
every
measurement= 0.2469,
n = 17 comparedand 26,) nor
in individualin male (p0.0011 (n26)) andfemale (p =(Figure
0.0049 4A,(n =p35))
participants,
withprior basal
subjects(Figure3B,D).
4B; n = 5, p = 0.3022).
values (Figure

Figure 3. Cortisol concentrations before and after a single group (cooking) or non-group (individual
Figure3. session.
Cortisol concentrations
beforeandfirst
after
a singleandgroup(cooking)
non-group(individual
activity)
CORT levels arefor the(before)second(after)orsaliva
samplescollected
activity)
CORT
areengaged
for the in
first(before)secondsalivasamplescollected
from fivesession.
men andnine levels
womencookingin andgroup
or in (after)
individualdeskor benchwork
fromfive men and ninein cooking Student’s
in group or
in individualdesk oractivity
bench workforfor approximately
1 h. womenp-valuesengaged
are for two-tailed
t-tests:(A) individual
by men,approximately
1 h.(B)p-values
two-tailed
Student’s
t-tests:(A) individual
activity
bypmen,p=p = 0.0087, n = 17;
cookingare
byfor
men,
p = 0.0011,
n = 26; (C)individual
activity by
women,= 0.2074,
0.0087,= 17;(B) cooking
by men,= 0.0011,= 26; (C) individual activity by women, p = 0.2074, n =n = 31; n(D)cookingby women,p = p0.0049,
n =n35.
31; (D) cooking by women, p = 0.0049, n = 35.

The percentage of the salivary CORT concentration changes was calculated by the values in
the first(before)averagesaliva overthe levels
secondin(after)
saliva saliva
samples,
as shown
in Figure
There samples
were noIn women,
CORT
the second
comparedwiththe first4. saliva
significant
differencesin after
CORT
concentration
in menduringboth forindividualand cooking
weresignificantly
lower
cooking
than in changes
individual
activity
sessions
every measurement
sessions4C;
as plottedin every(Figure 4A,= 0.2469, ndifference= 17 and 26,)
in individual(Figurep = 0.0385,
n = measurement
35 and 31). However,nopsignificant
wasnorobserved
when
subjects (Figure 4B; n = 5, p = 0.3022).

Behav. Sci. 2018, 8, x FOR PEER REVIEW

9 of 15compared for each individual (Figure 4D; p = 0.4498, n = 9). In addition, two-way ANOVA showedno significant interaction between marital status and sessions on CORT (F3,108 = 0.67, p = 0.5693).
Behav. Sci. 2018, 8, 101

9 of 15

Figure 4. Changes in cortisol levels before and after a single session. Percentage of CORT levels forthe first(before)saliva overthe second
samples
aftersession.
each session.
Salivaofwas
collected
Figure4. Changes
in cortisol
levels(after)
beforesaliva
and aftera single
Percentage
CORTlevels forat individual(non-cooking)and thegroup(cooking)
in menafter(A,B)each
andsession.
womenSaliva(C,D).wasAveragethe first (before)
saliva oversecond(after)activities
saliva samples
collected
percentage
of cortisol
levels forandthe group
first (before)
salivaover inthemensecondsaliva samplesin
at individual(non-cooking)(cooking)
activities(A,B)(after)
and women(C,D). Average
individual
participants.
p-values
two-tailed
Student’s
t-tests:= 0.2469(after)
betweenindividual
percentage
of cortisol
levelsare
forforthefirst (before)
salivaover (A)the psecond
salivasamples in
activity(n = 17) andcooking (np-values= 26) in men;
= 0.3022 between
individual
in 5
individual
participants.
are (B)
forp two-tailed
Student’s
t-tests:activity(A) pand= cooking0.2469 between
men;individual(C) p = 0.0385
betweenindividual
activity
(n
=
31)
and
cooking(n=35)
inwomen;(D)p=
0.4498activity (n = 17) and cooking (n = 26) in men; (B) p = 0.3022 between individual activity
betweenin 9between
women.individual
Filled and activity
open symbols
represent
marriedand individual
cooking in activity
5 men; and(C) cooking
p = 0.0385(n = 31)and cooking(n and= 35) insingleparticipants,
women;(D) p respectively.
= 0.4498 between individual activity and cooking in 9 women. Filled and open

symbols represent married and single participants, respectively.

In women, average CORT levels in the second saliva compared with the first saliva samples weresignificantly
lower after cooking than in individual activity sessions for every measurement (Figure 4C;
4. Discussion
p = 0.0385, n = 35 and 31). However, no significant difference was observed when compared forThis study
demonstrated
that
levelsin menbeforeANOVA
and after
cooking
were nearly
each individual(Figure4D; p = 0.4498,
n =OT9). Inaddition,
two-way
showedno significant
unchanged,
while
they
increased
duringnon-cooking
tasks.
Because
OT
levelsduringcooking
interaction between marital status and sessions on CORT (F3,108 = 0.67, p = 0.5693).
increased in women, we assume that OT reflects a happy condition [72–74]; these findings suggestthat cooking seems to be pleasant for women, but not so pleasant (actually rather stressful) for men.
4. Discussion
For women, cooking may not be extremely hard or stressful work, based on the lack of significant
This study demonstrated that OT levels in men before and after cooking were nearly unchanged,
differences between their choosing work (individual activity) and cooking.
while they increased during non-cooking tasks. Because OT levels during cooking increased in women,
We observed sex differences in OT concentration changes before and after cooking. However,
we assume that OT reflects a happy condition [72–74]; these findings suggest that cooking seems to
the differences were not dependent on marital status. Even though we inspected the data carefully,
be pleasant for women, but not so pleasant (actually rather stressful) for men. For women, cooking
no clear differences between married and single lifestyles were observed, though it is easily assumed
may not be extremely hard or stressful work, based on the lack of significant differences between theirthat cooking tasks in daily life are more familiar to married people. CORT concentrations after
choosing work (individual activity) and cooking.
cooking were significantly lower in both men and women, suggesting that group cooking seems not
We observed sex differences in OT concentration changes before and after cooking. However, theto be stressful for either sex.
differences were not dependent on marital status. Even though we inspected the data carefully, no
There is a circadian rhythm in CORT concentrations [75]; CORT concentrations are high in theclear differences between married and single lifestyles were observed, though it is easily assumed thatmorning, and gradually decrease in the afternoon. Therefore, it is not surprising that the CORT
cooking tasks in daily life are more familiar to married people. CORT concentrations after cooking
were significantly lower in both men and women, suggesting that group cooking seems not to be
stressful for either sex.

Behav. Sci. 2018, 8, 101

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There is a circadian rhythm in CORT concentrations [75]; CORT concentrations are high in themorning, and gradually decrease in the afternoon. Therefore, it is not surprising that the CORT
concentrations decreased after the 1-h period of each of the tasks. However, compared with thedecrease after individual activities, the decrease after cooking was much greater in both men andwomen, suggesting that the decrease following cooking was additive of the amount from circadianrhythms and was therefore actually due to the cooking activity.
There is a report that compared music and cooking interventions in a randomized controlled
trial of 48 patients with Alzheimer’s disease or mixed dementia [76]. Analyses revealed that bothmusic and cooking interventions led to positive changes in the patients’ emotional state and decreased
the severity of their behavioral disorders, as well as reduced caregiver distress. However, no benefit
on the cognitive status of the patients was seen. This suggests that cooking interventions may haveequivalent effects to music therapy. Unfortunately, they did not measure any biological markers
in either intervention. Because cooking interventions are effective in the emotional domain, OT
concentrations would have changed in their trials, likely similar to our results.
Group activities, usually with members who know each other or groups with fixed members,
have beneficial effects from a therapeutic viewpoint. In the psychology and psychiatry fields, grouptherapy is used extensively within public mental health services [77,78]. It has been shown thatcognitive therapy in groups improves the cognitive function of older people with dementia anddepression [65,79–81]. Group musical instrumental playing and choir singing are reported to improve
cognitive function, communication, impaired behavior, and mood [40,41,52–60], although the effects
are not always consistent [59].
On the relationship between group activities and OT concentration changes, there are two relevant
data. First, Kreutz [82] reported that a group of choir singers who singed together in a choir rehearsal
for half an hour and on another occasion the same individuals sit talking in small groups together. The
results showed that salivary OT increased after singing, with association of positive emotion increases
and negative emotion decreases, while equivalent changes were not observed after talking. Second, it
has been compared OT levels in amateur and professional singers during singing lessons [83]. Plasma
OT concentrations increased both in amateurs and professionals after the lesson in the group.
Quantifying OT and CORT concentrations in saliva is a non-invasive and user-friendly sampling
method [35,84,85] and is applicable to ASD subjects [41,67]. The precise relationship between OT
concentrations in CSF remains to be elucidated, but it has been shown that situations induce OT releasein the mouse brain more in the subordinate group than in the ordinate group according to social
stress [86]. Increases in saliva OT concentration is observed following social stress [35].
Salivary CORT is recognized as a biomarker of stress response in a protective mechanism
associated with acute and chronic stress [87,88] and CORT is released during stress [89]. CORT levels
after control and cooking activities decreased in the current tests. The detected levels (~100 ng/mL)
were identical to those reported for healthy adult males (88 ± 1.6 ng/mL; [90,91]). Kristenson et al. [92]reported that the correlation between CORT levels on consecutive 2- to 3-day periods was often in theorder of 0.5. For reliable CORT levels, mean levels over 2–3 days are used. Unfortunately, we did nottest in consecutive experiments; however, the CORT levels were the mean of several measurements indifferent days, which is more reliable than a single measurement.
Using saliva samples in this pilot trial was a non-invasive method that made it possible to study alarger group of participants in the future to confirm the current results. However, cooking interventions
are usually performed in small groups for practical reasons (e.g., a group using a kitchen in a nursinghome), which makes it difficult to perform a large-scale cohort.
This study did not address emotional changes after cooking. Such tests should be performed
using a questionnaire survey. It is expected that the cooking was a pleasant experience because theproducts (mostly pickles, Supplementary Figure S1) from the cooking session were shared with theparticipants after the 3–10 days of pickling with salt or vinegar. In other words, all the participants
knew they were to be rewarded with the products of their cooking.

Behav. Sci. 2018, 8, 101

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The investigation had some limitations. Though the salivary OT and CORT levels seem to havebeen affected, the menstrual cycle in female participants was not considered and sex hormone levels
were not evaluated.
During the 3 years of this study, the participants were not divided into fixed groups and different
numbers of participants joined each year. However, the participants were all acquainted and sevenmembers of our laboratory started and ended the study together. It is not surprising that the groups
were so fluid because many behavioral treatments in the community take place in open groups or arolling format where group members join at different times and membership changes, as current study.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-328X/8/11/101/
s1, Figure S1: Cooking in a group performed by students from the medical school and laboratory workers. Figure
S2: Salivary OT levels in adult samples after intestinal delivery of different concentrations of OT.
Author Contributions: H.H., T.Y. and K.F. designed the experiments. H.H. and T.Y. wrote the manuscript. T.Y.,
K.I. (Kosuke Ise), K.I. (Kei Iwashina), N.T., S.Y., K.S., T.M., A.Y., K.K., and K.F. measured OT concentrations.
S.M.C., M.G., A.A.S., K.H., H.K., and H.Z. performed the cooking experiments. C.T. and S.Y. gave advises during
students’ experiments.
Funding: This work was supported by grant-in-aid from “Integrated research on neuropsychiatric disorders”
carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Conflicts of Interest: The authors declare no conflict of interest.

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