The effects of laughter on attention focusing and psychological stress in healthy older adults: a single-blind, randomized controlled trial using a comic video intervention

Abstract

Background
Disorders associated with mental health significantly impact disability-adjusted life year values and represent a social problem in stressed societies. Worsening mental health also affects cognitive functions and quality of life. To address these issues, increasing attention is attracted to preventive measures vis-à-vis mental and brain health in daily life. Therefore, growing interest in care using laughter has recently been noted. This study was designed to assess the effects of a short-term laughter-based intervention on the mental health and cognitive functions of middle-aged and older adults.

Methods
The study applied a single-blind, crossover-controlled trial design. Cognitive tasks were performed after participants viewed a video clip of approximately four minutes (humor or control video) and the resulting scores were treated as the primary endpoint. Secondary endpoints included cerebral blood flow in the dorsolateral prefrontal cortex, heart rate variability, subjective mood state assessment, and salivary stress biomarkers.

Results
The study was conducted on 25 healthy Japanese-speaking adults aged 40 to 65 years. Results revealed a significant increase in digit vigilance scores and in comparison to viewing the control video, participants evinced a trend toward an increase in serial 7 subtraction scores after viewing the humor video. No significant differences were found in scores on other cognitive tasks. The cerebral blood flow was also significantly higher in the dorsolateral prefrontal cortex during cognitive tasks performed after participants viewed the humor video compared to the control video. The outcomes of heart rate variability, subjective mood state assessment, and salivary stress markers also suggested that the humor video intervention could subsequently contribute to the activation of parasympathetic activity and reduce psychological stress levels induced by the cognitive tasks.

Conclusions
The study outcomes indicated that interventions using short humor videos can improve attention focus and may help to reduce psychological stress levels. These results support the clinical benefits of humor, which could be utilized as a simple and non-invasive approach to promoting the health of middle-aged and older adults.

Trial registration
The study was registered at the University Hospital Medical Information Network (UMIN) database (Registration No. UMIN000043332||http://www.umin.ac.jp/ctr/) on 15/02/2021.

Full text

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The effects of laughter on attention focusing and psychological
stress in healthy older adults: a single-blind, randomized controlled
trial using a comic video intervention
Tatsuya Yamakoshi
Kirin Central Institute, Kirin Holdings Company
Ryo Sakamoto
Sakai City Medical Center
Takafumi Fukuda
Kirin Central Institute, Kirin Holdings Company
Ayana Kanatome
Kirin Central Institute, Kirin Holdings Company
Atsuko Koyama
Shiroyama Hospital
Yasuhisa Ano
Kirin Central Institute, Kirin Holdings Company
Research Article
Keywords: Laughter, Humor, Focusing attention, Cerebral blood ow, Psychological stress levels, Parasympathetic activity, Mental health,
Well-being
Posted Date: July 12th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4598246/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License
Additional Declarations: No competing interests reported.

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Abstract
Background
Disorders associated with mental health signicantly impact disability-adjusted life year values and represent a social problem in stressed
societies. Worsening mental health also affects cognitive functions and quality of life. To address these issues, increasing attention is
attracted to preventive measures vis-à-vis mental and brain health in daily life. Therefore, growing interest in care using laughter has
recently been noted. This study was designed to assess the effects of a short-term laughter-based intervention on the mental health and
cognitive functions of middle-aged and older adults.
Methods
The study applied a single-blind, crossover-controlled trial design. Cognitive tasks were performed after participants viewed a video clip of
approximately four minutes (humor or control video) and the resulting scores were treated as the primary endpoint. Secondary endpoints
included cerebral blood ow in the dorsolateral prefrontal cortex, heart rate variability, subjective mood state assessment, and salivary
stress biomarkers.
Results
The study was conducted on 25 healthy Japanese-speaking adults aged 40 to 65 years. Results revealed a signicant increase in digit
vigilance scores and in comparison to viewing the control video, participants evinced a trend toward an increase in serial 7 subtraction
scores after viewing the humor video. No signicant differences were found in scores on other cognitive tasks. The cerebral blood ow
was also signicantly higher in the dorsolateral prefrontal cortex during cognitive tasks performed after participants viewed the humor
video compared to the control video. The outcomes of heart rate variability, subjective mood state assessment, and salivary stress
markers also suggested that the humor video intervention could subsequently contribute to the activation of parasympathetic activity and
reduce psychological stress levels induced by the cognitive tasks.
Conclusions
The study outcomes indicated that interventions using short humor videos can improve attention focus and may help to reduce
psychological stress levels. These results support the clinical benets of humor, which could be utilized as a simple and non-invasive
approach to promoting the health of middle-aged and older adults.
Trial registration
The study was registered at the University Hospital Medical Information Network (UMIN) database (Registration No.
UMIN000043332||http://www.umin.ac.jp/ctr/) on 15/02/2021.
Background
Intense competition, along with high-pressured lifestyles and social conditions adversely impact individuals living in present-day societies,
causing stress and poor quality of life, which are becoming urgent social problems [1, 2]. Mental health exerts a signicant impact on
disability-adjusted life year values [3]. The deterioration of mental health is also associated with cognitive decline and signicantly
impacts labor productivity [4, 5]. Chronic mental health diculties can trigger mood disorders such as anxiety and depression; hence,
preventive care is essential for the resolution of problems before they become severe. Given this context, a growing interest in care using
laughter has been observed in recent years [6]. Laughter therapy does not require specialized equipment and high costs and has thus
already been introduced in the medical domain as a treatment program without side effects [7, 8]. An observational study conducted on
14,233 individuals demonstrated an association between infrequent laughter and a higher risk of developing a functional disability [9].
Laughter also contributes to psychosocial and physical health aspects and several studies [10, 11, 12, 13, 14, 15] have shown that it can
effectively alleviate negative emotions such as anxiety and depression. Physiologically, laughter reduces stress hormones [6, 14] and
increases T lymphocytes by activating natural killer cells, which causes an upsurge in white blood cells and immunoglobulin (Ig) levels,
thus increasing a body’s immunity [15, 16, 17]. Laughter also lowers blood pressure: it reduces vasoconstriction through a decrease in the

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breakdown of nitric oxide, a vasorelaxant substance, and raises blood glucose levels by reducing cortisol [14, 18, 19]. Laughter therapy is
non-invasive, non-pharmacological, and relatively easy to implement; it is thus considered an effective complementary treatment that can
reduce the intensity of many diseases [20] and is also expected to ameliorate the health of middle-aged and older adults [21]. The effects
of laughter have been demonstrated on a range of psychosocial and physical health outcomes as noted above; however, the extant
research has not comprehensively tested how positive psychological states achieved from short-term laughter inuence cognitive
functioning and stress responses. Previous studies [22] have shown that humor video interventions improve short-term memory in healthy
older people, suggesting that such means could improve cognitive functions. However, the effects of humor video interventions on other
cognitive functions (such as attention focusing and working memory) or on changes in the biological responses of middle-aged people
remain unclear. The present study designed an intervention trial with middle-aged and older adults to assess the effects of a brief
laughter-based intervention on their autonomic nervous systems and stress responses and to determine the effects of autonomic
improvement on cognitive functions, primarily attention. We hypothesized that laughter interventions would be associated with improved
cognitive functions and increased regional cerebral blood ow (rCBF) in the dorsolateral prefrontal cortex (DLPFC) during the performance
of cognitive tasks. We used near-infrared spectroscopy (NIRS) to measure rCBF during the intervention. The DLPFC region of the
prefrontal cortex enacts an essential role in the executive functions of working cognition exibility [23], planning [24], memory, inhibition,
and reasoning; it is also crucial for the executive functions of cognitive process management [25]. In addition, we measured changes in
salivary stress biomarkers [26, 27, 28, 29, 20, 31, 32] (cortisol, α-amylase, s-IgA, and β-endorphin) at every given event and calculated the
autonomic nervous system activity using heart rate variability (HRV) [33] as biometric information associated with mood state.
Methods
Ethics and registration
This study was conducted following the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human
Subjects and was approved by the ethics committee of Kirin Holdings Company. All participants provided written informed consent.
Before enrolling participants, the study was registered at the University Hospital Medical Information Network (UMIN) database
(Registration No. UMIN000043332||http://www.umin.ac.jp/ctr/; Registration title. Effects of watching videos on brain function: A single-
blind, crossover comparative study) on 15/02/2021.
Participants
We recruited 25 healthy middle-aged and older Japanese-speaking adults aged between 40 and 65 years. In a previous study[22], the
effect of humor on short-term memory were reported in 20 healthy older adults. Based on this report, an equivalent sample size was
established in this study. The following exclusion criteria were established:
(i) Suffering from hearing or visual impairment that interfered with daily life
(ii)Wearing a device that interfered with heart rate measurement (e.g., pacemaker)
(iii) Having developed symptoms of arrhythmia in the past 12 months
Intervention
Experimental intervention
The experimental intervention required participants to view a suciently funny comic dialog video (humor video) of approximately four
minutes. The audiovisual clip was provided by Yoshimoto Kogyo Holdings Company (Tokyo, Japan).
Control intervention
The control intervention made participants view a less funny, approximately four-minute-long comic dialog video clip (control video)
provided by Yoshimoto Kogyo Holdings Company (Tokyo, Japan).
After viewing the video clips, the participants rated them on a questionnaire comprising seven items: funniness, frequency of laughter,
understanding, sympathy, exciting language, enjoyable movement, and tempo.
Procedure
A randomized, single-blind, crossover comparative design was applied for the trial. Registered participants were alternately allocated to
two sequences (1 and 2) in the order of their registration to exclude the order effect reective of the experiment’s experience. Participants

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designated to sequence 1 viewed the humor video as the experimental intervention in Phase 1 and the control video as the control
intervention in Phase 2. The order was reversed for participants assigned to sequence 2.
The interval between Phase 1 and Phase 2 was > 15 minutes. Figure1 presents the examination ow on the study day.
The study sequence allocator was not involved in assessing eligibility, data collection, or analysis. The research staff and outcome
assessors were blinded to sequence allocations until data analysis. On the trial day, the participants were instructed to avoid caffeine-
containing foods and beverages, excessive exercise, alcohol consumption, and smoking.
The participants were also instructed not to eat or drink anything except water for two hours before the intervention. The data were
collected in April 2020 at Hamamatsu City in Shizuoka prefecture in Japan. This study was conducted by Kirin Holdings Company (Tokyo,
Japan).
Primary outcomes
The primary outcome comprised the scores of the cognitive tasks performed by the participants.
Participants took a ve-minute rest after viewing the humor or control video as previously described. They subsequently performed the
cognitive tasks comprising digit vigilance, serial 7 subtraction, alphabetic working memory, and n-back (Fig.1). The participants were
granted a 20-second rest period for each task. The digit vigilance, serial 7 subtraction, and alphabetic working memory tasks were
presented using the Computerized Mental Performance Assessment System (COMPASS, Northumbria University, Newcastle upon Tyne,
UK), a purpose-designed software application for the exible delivery of randomly generated parallel versions of standard and novel
cognitive assessment tasks. This assessment system has also been previously utilized to measure the effectiveness of nutritional
interventions [35, 36].
Fixed numbers were displayed on the right side of the screen for the digit vigilance task, and numbers changing at the rate of 150 per
minute were presented one after another on the left side of the screen. Participants were required to respond when the number on the left
matched the number on the right. The task lasted for 1.5 minutes and the participants were scored for accuracy (%), reaction times for
correct responses (ms), and false alarms (number).
A random number between 800 and 999 was presented on the screen for the serial 7 subtraction task and participants were required to
continually subtract seven from the presented number as quickly as possible. The starting number was cleared on the entry of the rst
answer, and asterisks indicated the next three digits. This task was scored for the number of responses (number), the number of correct
responses (number), and false alarms (number).
The alphabetic working memory task displayed a series of ve letters on the screen, one letter at a time, and participants memorized the
displayed letters. Thirty letters were then displayed on the screen one by one and participants were required to press computer keyboard
keys corresponding to YES or NO as quickly as possible to answer whether the letter belonged to the original series. The task was scored
for accuracy (%) and reaction times for correct responses (ms).
The n-back task was presented on a touchpad using brain training apps (Kirin Holdings Company, Japan). The n-back task required
participants to memorize the colors and shapes of trains displayed one after the other. Subsequently, participants were asked to tap the
train whose color and shape corresponded to the one displayed three trains before. This task was scored for accuracy (%), the number of
correct responses (number), and false alarms (number).
Secondary outcomes
Measurements of cerebral blood ow
The rCBF was measured during the intervention using a 2CH NIRS system (HOT-2000, NeU Corporation, Tokyo, Japan) with a single
wavelength of 810 nm, and the concentration change in total hemoglobin (total Hb) was calculated as demonstrated in a previous report
[37] (Fig.1).
Two dual-source detectors were placed in the left and right DLPFC. This area corresponds to the Fp1-Fp2 region as dened by the
international 10–20 system used in electroencephalography [38, 39]. Total Hb was dened as the mean value of Hb in the left and right
DLPFC. The pre-task baseline was dened as the mean value of total Hb in the 60 seconds before beginning the cognitive tasks. The
mean values of total Hb during the task period minus the baseline were compared.

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Measurement of HRV
ECG was measured using a Silmee Bar type Lite (TDK Corporation, Tokyo, Japan) at a time resolution of 1000 Hz [40]. The participants
were asked to apply the device to the anterior chest with a gel pad. ECG measurements were taken from before the start of the
intervention until after the end of the cognitive tasks (Fig.1). The RR interval was calculated from the ECG data by the Slimee
measurement and analysis system, and HRV analysis was then performed using Kubios HRV analysis software ver 3.4.1. A threshold-
based artifact correction algorithm was employed to correct artifacts and ectopic beats in the RR interval data HRV parameters in the
frequency domain, specically LF (0.04–0.15 Hz), HF (0.15–0.4 Hz), the ratio between LF and HF band powers (LF/HF), and total power
(TP, 0–0.4 Hz) were analyzed using FFT. HF was used as the indicator of parasympathetic activity [41], LF/HF as the marker for
sympathetic activity [42], and TP as the sign of the overall autonomic nervous system activity [41].
Assessment of the subjective mood state of participants
The visual analog scale (VAS) and TDMS-ST [43] were used to assess the mood states of participants. The VAS evaluated fatigue, stress,
motivation, depression, concentration, and drowsiness of participants, who rated their current mood state on 100-mm VAS before and
after the video intervention and after performing the cognitive tasks (Fig.1). The VAS scores were converted to z-scores for each
participant to reduce the inuence of subjective rating scales and to ensure appropriate comparisons. The z-scores were calculated using
the mean and standard deviation changes in the VAS scores of every participant. Consequently, the mean and the standard deviation were
adjusted to a z-score of 0 and 1 for each participant.
The TDMS-ST comprises eight mood descriptors: energetic, lively, lethargic, relaxed, calm, irritable, and nervous. The participants reported
their current mood by rating each item on a six-point Likert-like scale before and after the video intervention, and after completing the
cognitive tasks (Fig.1). Vitality, stability, pleasure, and arousal scores were then calculated according to the established protocol.
Salivary biomarkers
Salivary cortisol, α-amylase, s-IgA, and β-endorphin were measured as stress biomarkers, and salivary oxytocin was calculated as the
indicator of psychological fulllment. Participants were instructed to collect drooling saliva using a saliva collection aid (SalivaBio
Corporation, Carlsbad, CA, United States) before and after the video intervention and after accomplishing the cognitive tasks (Fig.1). The
collected saliva samples were measured at the Yanaihara Institute Inc. (Shizuoka, Japan).
Statistical analysis
Data were expressed as mean ± standard deviation. Comparisons between the experimental and control phases were accomplished using
paired t-tests. The Sidak test was applied to compare temporal changes during the same phase. Statistical comparisons were performed
using BellCurve for Excel (Social Survey Research Information, Tokyo, Japan) and Microsoft Excel 2016 (Microsoft, Redmond, WA, USA).
Statistical signicance was set at
p
< 0.05.
Missing values for each outcome were dened as meeting the following criteria.
Score for cognitive tasks; Data from participants who self-reported that they gave up performing the task.
Measurements of cerebral blood ow; Data from subjects who self-reported that they gave up performing the task.
Measurement of HRV; Data evincing a data loss rate of more than 50% due to poor ground contact between the sensor and skin.
Assessment of the subjective mood state of participants; Non-response data.
Salivary biomarkers; Data below detection limit.
Results
Baseline characteristics of the study sequences
Figure 2 presents the study owchart. We recruited 25 participants (male/female = 12/13). Registered participants were alternately
allocated as previously mentioned: Sequence 1 comprised 13 participants and sequence 2 encompassed 12 participants. Alternate
assignments were made in each sequence for male and female participants to avoid gender bias. All participants completed the study;
therefore, the total analyzed data included all 25 participants, whose characteristics are displayed in Table1. There were no signicant
between-sequences differences in baseline characteristics. No serious adverse effects were observed.

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Table 1
Subject characteristics at baseline
Characteristics Sequence1 (n = 13) Sequence2 (n = 12)
p
-value
Age 49.5 ± 6.7 52.8 ± 6.9 0.25
Male / Female 6 / 7 6 / 6 0.74
Data were presented as means standard deviations (SD);
p
-value of the age was calculated using unpaired t-tests. The sex ratio was
analyzed using the χ2 tests.
Evaluation of video clips
Table2 shows the participant ratings reported for each video after it was viewed. The humor video was rated signicantly higher than the
control video on all assessment items (funniness, frequency of laughter, understanding, sympathy, exciting language, enjoyable
movement, and tempo) (
p
< 0.001, all assessment items). This result conrmed that the humor video was appropriately funny in
comparison to the control video.
Table 2
Evaluation of video clips
Evaluation item Humor video Control video
n p
-value
Funniness 5.9 ± 0.3 2.2 ± 0.2 25 p < 0.001
Frequency of laughter 4.0 ± 0.2 1.6 ± 0.1 25 p < 0.001
Understanding 6.5 ± 0.2 3.4 ± 0.4 25 p < 0.001
Sympathy 5.8 ± 0.3 2.6 ± 0.3 25 p < 0.001
Exciting language 6.2 ± 0.2 2.7 ± 0.4 25 p < 0.001
Enjoyable movement 5.3 ± 0.3 2.4 ± 0.2 25 p < 0.001
Tempo 6.5 ± 0.1 3.6 ± 0.4 25 p < 0.001
Data were presented as means standard error (SE);
p
-value was calculated using paired t-tests
Primary outcomes
Scores for cognitive tasks
Table3 records the scores for the cognitive tasks performed after the viewing of each video (humor and control). Response speeds for
the digit vigilance tasks accomplished after viewing each video were signicantly faster for the humor video than for the control video (
p
=
0.039). Similarly, the number of correct responses for the serial 7 subtraction tasks undertaken after participants viewed each video
tended to be higher for the humor video (
p
= 0.079). No signicant differences were found in the other scores. However, participants
performed better on many scores after viewing the humor video than after viewing the control video.

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Table 3
Score for the cognitive tasks
Cognitive task Humor video Control video
n p
-value
Digit vigilance Accuracy (%) 97.39 ± 0.69 97.54 ± 0.67 23 0.87
Reaction times for correct responses (ms) 462.78 ± 6.81 473.00 ± 6.64 23 0.039
False alarms (number) 0.74 ± 0.20 0.83 ± 0.21 23 0.70
Serial 7 subtraction The number of responses (number) 21.24 ± 1.63 20.33 ± 1.42 21 0.26
The number of correct responses (number) 19.10 ± 1.51 17.57 ± 1.42 21 0.079
False alarms (number). 2.14 ± 0.42 2.76 ± 0.44 21 0.30
Alphabetic working memory Accuracy (%) 94.49 ± 1.25 94.30 ± 1.25 23 0.88
Reaction times for correct responses (ms) 984.96 ± 30.88 983.18 ± 32.40 23 0.94
N-back Accuracy (%) 87.87 ± 2.6 87.19 ± 2.48 21 0.79
The number of correct responses (number) 12.30 ± 0.36 12.21 ± 0.35 21 0.79
False alarms (number) 1.70 ± 0.36 1.79 ± 0.35 21 0.79
Data were presented as means standard error (SE);
p
-value of the age was calculated using paired t-tests.
Table3. Score for the cognitive tasks
Secondary outcomes
Measurements of cerebral blood ow
We measured the changes from the pre-task baseline in total Hb concentration during the cognitive tasks. These results are presented in
Tables4 and 5. There was no signicant difference at baseline before the intervention. Compared to the pre-task baseline, the total Hb
concentration of participants increased signicantly during the serial 7 subtraction, the alphabetic working memory, and the n-back tasks
performed after viewing the humor video (
p
< 0.001,
p
= 0.0014,
p
< 0.001, respectively). In contrast, no signicant change was detected
compared to the pre-task baseline when these tasks were performed after viewing the control video. Further, the changes in total Hb
concentrations while undertaking the cognitive tasks were signicantly greater than the pre-task baselines after participants had viewed
the humor video compared to after they had viewed the control video. (
p
= 0.022; Fig.3 and Table5)
Table 4
The changes from the pre-task baseline in total Hb concentration
Cognitive task Hb concentration
n p
-value
After viewing the humor video Digit vigilance 0.08 ± 0.04 23 0.73
Serial 7 subtraction 0.28 ± 0.06 23 p < 0.001
Alphabetic working memory 0.18 ± 0.05 23 0.0014
N-back 0.23 ± 0.07 23 p < 0.001
During all tasks 0.18 ± 0.05 23 p < 0.001
After viewing the control video Digit vigilance 0.03 ± 0.06 23 1.00
Serial 7 subtraction 0.11 ± 0.06 23 0.064
Alphabetic working memory 0.06 ± 0.05 23 0.87
N-back 0.08 ± 0.05 23 0.39
During all tasks 0.07 ± 0.05 23 0.74
Data were presented as means standard error (SE);
p
-value was calculated using sidak test.

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Table 5
Comparison of the changes from the pre-task baseline in total Hb concentration
After viewing the humor video After viewing the control video
n p
-value
Digit vigilance 0.08 ± 0.04 0.03 ± 0.06 23 0.25
Serial 7 subtraction 0.28 ± 0.06 0.11 ± 0.06 23 0.014
Alphabetic working memory 0.18 ± 0.05 0.06 ± 0.05 23 0.031
N-back 0.23 ± 0.07 0.08 ± 0.05 23 0.023
During all tasks 0.23 ± 0.07 0.07 ± 0.05 23 0.022
Data were presented as means standard error (SE);
p
-value was calculated using paired t-tests.
Measurement of HRV
Supplementary Tables1 and 2 show the time series changes in the actual measured HRV values. LF/HF was signicantly higher when the
humor video was viewed compared to when participants viewed the control video (
p
= 0.012). Conversely, the LF/HF measured during the
cognitive tasks was lower after viewing the humor video compared to after participants viewed the control video. A statistically signicant
difference was noted during the performance of the last task (N-back). (
p
= 0.002) (Fig.4). Time series changes in HF exhibited an inverse
change to the changes noted in LF/HF. No signicant changes were observed in TP (Supplementary Tables1 and 2).
Subjective mood state assessment by participants
VAS z-scores for fatigue, stress, motivation, depression, and concentration were signicantly higher (
p
= 0.0012,
p
< 0.001,
p
< 0.001,
p
=
0.0031,
p
= 0.0032, respectively; Table6) after viewing the humor video compared to after the control video was viewed. Further, the
scores allotted by participants for stress, motivation, and depression after completing the cognitive tasks were signicantly higher after
viewing the humor video compared to after they had viewed the control video (
p
= 0.0027,
p
= 0.0021,
p
= 0.0083 respectively; Table6).
However, no signicant differences were observed in the drowsiness scores. No mood state was signicantly different at baseline before
the intervention. The actual scores of vitality, stability, and pleasure were signicantly higher in TDMS-ST after participants had viewed the
humor video compared to after they had viewed the control video (
p
= 0.0017,
p
= 0.013; Supplementary Table3). No signicant
differences were observed in arousal scores.

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Table 6
Score for VAS
Mood states Humor video Control video
n p
-value
Fatigue Before the video intervention -0.04 ± 0.17 -0.02 ± 0.10 24 0.78
After the video intervention 0.79 ± 0.12 -0.05 ± 0.17 24 0.0012
After performing the cognitive tasks -0.49 ± 0.19 -0.87 ± 0.16 24 0.15
Stress Before the video intervention -0.03 ± 0.15 -0.08 ± 0.12 24 0.093
After the video intervention 0.86 ± 0.11 0.03 ± 0.15 24 p < 0.001
After performing the cognitive tasks -0.42 ± 0.15 -1.13 ± 0.14 24 0.0027
Motivation Before the video intervention 0.02 ± 0.16 -0.15 ± 0.17 24 0.75
After the video intervention 0.64 ± 0.12 -0.25 ± 0.16 24 p < 0.001
After performing the cognitive tasks 0.12 ± 0.17 -0.95 ± 0.16 24 0.0021
Depression Before the video intervention 0.04 ± 0.16 0.03 ± 0.17 24 0.093
After the video intervention 0.65 ± 0.14 -0.01 ± 0.15 24 0.0031
After performing the cognitive tasks -0.32 ± 0.13 -0.95 ± 0.16 24 0.0083
Concentration Before the video intervention 0.26 ± 0.13 -0.13 ± 0.19 24 0.18
After the video intervention 0.38 ± 0.15 -0.38 ± 0.16 24 0.0032
After performing the cognitive tasks 0.13 ± 0.17 -0.43 ± 0.25 24 0.058
Drowsiness Before the video intervention 0.12 ± 0.15 0.00 ± 0.22 24 0.75
After the video intervention 0.17 ± 0.17 -0.20 ± 0.14 242 0.14
After performing the cognitive tasks 0.06 ± 0.19 -0.42 ± 0.20 24 0.069
Data were presented as means standard error (SE);
p
-value of the age was calculated using paired t-tests.
Table6. Score for VAS
Salivary biomarkers
Supplementary Table4 displays the salivary cortisol, α-amylase, s-IgA, β-endorphin, and oxytocin measurements before and after
participants had viewed each video, and after the cognitive tasks, respectively. No signicant differences were observed between the
experimental and control interventions. The changes in α-Amylase from before viewing the video to after the cognitive tasks tended to be
higher after viewing the control video compared to after viewing the humor video (
p
= 0.060; Supplementary Table4). No differences
between the two interventions were noted in the changes in cortisol, s-IgA, β-endorphin, and oxytocin.
Discussion
This single-blind, crossover, and comparative study recruited 25 healthy adults aged between 40 and 65 to investigate how a short humor
video intervention affected cognitive functions and mood states. The study utilized the digit vigilance task to assess focusing attention
[44, 45] and employed the serial 7 subtraction, alphabetic working memory, and n-back tasks to assess working memory [46, 47, 48]. The
results revealed that the digit vigilance scores improved signicantly and that the serial 7 subtraction scores tended to increase after the
humor video intervention compared to after the control video was shown. It is therefore suggested that viewing short humor videos could
enhance attention focus. A previous study [22] found that viewing humor videos improved short-term memory in healthy older people, but
the current result is the rst report on attention focusing. In addition, the present study evidenced the effects of video-based interventions
on middle-aged and older adults (40–65 years), whereas previous investigations [22] have targeted people aged 65 and older.
Noteworthily, while previous studies [22, 49] have used 20-minute humor videos, the current study conrmed the constructive effects of
viewing even shorter funny video clips of only four minutes. A further feature of this study involved the real-time measurement of the
biometric impact of the short humor video intervention using a NIRS[40, 50]. The rCBF in the DLPFC region was signicantly higher during

Page 10/17
the cognitive tasks after participants had viewed the humor video compared to after they had viewed the control video. The DLPFC region
of the prefrontal cortex enacts an essential role in the executive functions of cognitive process management [25]. Extant research has
shown that damage to the DLPFC impairs attention focusing [51]. DLPFC dysfunction has also been associated with decreased attention
focus, increased mental health problems [52], and reduced rCBF in the DLPFC. The outcomes of this study suggest that the viewing of
short humor videos promotes DLPFC activity during cognitive tasks and is associated with improvements in DLPFC-related cognitive
functions such as attention focusing and may also exert constructive effects vis-à-vis problems related to mental health.
The VAS and TDMS-ST scores were used to assess subjective mood states and were signicantly higher for all categories except
sleepiness and arousal after participants had viewed the humor video compared to after they had viewed the control video. As previous
studies have described [12, 13], laughter was found to effect temporary improvements in mood state. LF/HF is an indicator of sympathetic
activity, and HF is an indicator of parasympathetic activity [33, 53, 54]. LF/HF rose transiently while watching the humor video and then
quickly declined. HF was signicantly higher during the last cognitive task after participants had viewed the humor video compared to
after they had viewed the control video. Laughter may have stimulated a transient activation of sympathetic activity, which may have
increased subsequent parasympathetic activity under the inuence of homeostasis. Parasympathetic activity has been reported to be
associated with reduced psychological stress[54], suggesting that laughter reduces psychological stress. At the same time, the changes
in VAS scores registered after the cognitive tasks for stress, depressed mood, and motivation were signicantly higher after participants
had viewed the humor video compared to after they had viewed the control video. These results suggest that the humor video intervention
could have contributed subsequently to the activation of parasympathetic nerve and reduced the psychological stress levels induced by
the cognitive task.
Further, changes in α-amylase activity from before the videos were viewed to after the cognitive tasks were accomplished tended to be
higher after participants had viewed the control video compared to after they had viewed the humor video. The α-amylase activity is a
known indicator of physical and mental stress [55] and is known to respond on a relatively fast timescale. The results of this study
suggested that the humor video intervention may have diminished the increase in stress levels during the tests. Unlike previous studies [6,
14, 18], the current investigation observed no changes in cortisol concentrations. This result could be attributed to the relative brevity of
the humor video, which was only around four minutes and may not have allowed enough response time for these indicators to change.
These results support the contention that the viewing of short humor videos by middle-aged and older adults could promote DLPFC
activity and thus enhance cognitive functions such as attention focus and reduce psychological stress levels through the subsequent
activation of parasympathetic activity.
The study must acknowledge several limitations. First, the sample size was small. The study’s sample had adequate power as conrmed
by the results of previous studies; however, a larger sample size would have allowed a better assessment of the middle-aged and older
adults population as a whole. Second, we did not measure the effects of repeated interventions using the humor video. Third, we only
measured the immediate post-intervention effects of the humor video and therefore did not evaluate how long the effects of laughter
would last. Future initiatives would need to demonstrate the effectiveness of repeated interventions and the persistence of the effects for
laughter to be routinely and continuously utilized as a tool for the amelioration of cognitive functions and the alleviation of stress levels in
the daily lives of individuals.
Conclusions
This study aimed to determine the impact of a short humor video intervention on the cognitive functions, mood states, and associated
biometric information of the participants. The study outcomes indicated that the conducted intervention using a short humor video clip
increased rCBF during the cognitive tasks and may have improved attention focus. The results of the study also suggested that
interventions involving short humor videos could be associated with reduced psychological stress levels. These enjoyable interventions
can represent continuous and practicable approaches to diculties of daily living and could contribute to improvements in the cognitive
functions and stress levels of middle-aged and older adults.
List Of Abbreviations
rCBF regional cerebral blood ow
DLPFC The dorsolateral prefrontal cortex
NIRS near-infrared spectroscopy

Page 11/17
HRV heart rate variability
UMIN university hospital medical information network
COMPASS computerized mental performance assessment system
VAS visual analog scale
Declarations
Ethics approval and consent to participate
This study was conducted following the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human
Subjects and approved by the ethics committee of Kirin Holdings Company. All participants provided written informed consent. The study
was registered at the UMIN database before subject enrollment (Registration No. UMIN000043332||http://www.umin.ac.jp/ctr/;
Registration title. Effects of viewing videos on brain function: A single-blind, crossover comparative study) on 15/02/2021.
Consent for publication
Not applicable.
Availability of data and materials
The data presented in this study are available from the corresponding author upon reasonable request
Competing interests
This research was conducted with the support of a research fund from Kirin Holdings Company, Ltd.
Funding
This study was funded by Kirin Holdings Company, Ltd.
Authors’ contributions
T.Y., Y.A., and A.K. designed the study, posited the principal conceptual ideas, and wrote the draft outline. T.Y., Y.A., and T.F. collected the
data. A.K. and R.S. contributed to the interpretation of the results. Y.T. wrote the manuscript with support from Y.A. All authors have read
and approved the nal manuscript.
Acknowledgements
We appreciate Mr. Kuniaki Obara for his assistance in managing this clinical project.
We acknowledge the Hamamatsu City Hall ocers involved in this study and thank the citizens of Hamamatsu for participating in our
study. We are also grateful to Yoshimoto Kogyo Holdings Company for providing us with the intervention videos.
Author information
1Kirin Central Institute, Kirin Holdings Company, Limited, 2-26-1, Muraoka-higashi, Fujisawa-shi, Kanagawa, 251-8555, Japan.
2Department of Psychosomatic Medicine, Kindai University Faculty of Medicine, Osakasayama, Osaka, Japan
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Figures
Figure 1
Measurement Flow
Participants performed four Cognitive tasks after viewing the video. Assessment of the subjective mood state and Drooling saliva
collection was performed in (1), (2), and (3) shown in the gure. rCBF and HRV were measured throughout the study.

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Figure 2
CONSORT diagram
All 25 participants recruited met the entry requirements. Participants were assigned alternately to Sequence 1 (n=13) and Sequence 2
(n=12). All participants were included in the analysis.

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Figure 3
Measurement of cerebral blood ow
The total Hb concentration was measured using 2CH NIRS. The mean values of total Hb concentration during all task periods minus the
baseline were shown. Data were represented as means ± standard error (SE) for after viewing the control video and after viewing the
humor video. Group differences were identied by paired t-tests; *
p
< 0.05.

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Figure 4
Measurements of LF/HF
LF/HF was calculated by ECG, and ECG was measured using Silmee Bar type Lite. The time ow of LF/HF in the phase of the humor video
(solid line) and the phase of the control video (dashed line) was shown. Data were represented as means ± standard error (SE). Group
differences were identied by paired t-tests; *
p
< 0.05, **
p
< 0.001.
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