Health, 2018, 10, 1159-1170
ISSN Online: 1949-5005
ISSN Print: 1949-4998
10.4236/health.2018.109088 Sep. 6, 2018 1159 Health
Effect of 528 Hz Music on the Endocrine System
and Autonomic Nervous System
Kaho Akimoto1, Ailing Hu2, Takuji Yamaguchi2, Hiroyuki Kobayashi1,2
1Department of Hospital Administration, Graduate School of Medicine, Juntendo University, Tokyo, Japan
2Center for Advanced Kampo Medicine and Clinical Research, Graduate School of Medicine, Juntendo University, Tokyo, Japan
This study examined the stress-
reducing effect on the endocrine system and
nervous system of music with a frequency of 528 Hz, which
has recently attracted attention as a “healing” type of music.
participants (one man and eight women, aged 26 -
37 years) listened to 528
Hz and standard 440 Hz music on separate days. We measured salivary bio-
markers of stress (cortisol, chromogranin A, and oxytocin
) before and after
exposure to music, and continuously recorded the activity of the autonomic
nervous system. The Profile of Mood State, 2nd edition, was also administered
as a subjective indicator of stress. In the 528 Hz condition, mean levels of cor-
tisol significantly decreased, chromogranin A tended to decrease, and oxyto-
cin significantly increased after music exposure. However, no signifi
change was observed in any salivary biomarkers in the 440 Hz condition. The
ratio of low frequency to high frequency autonomic nervous system activity
significantly decreased after exposure to both types of music, and the coeffi-
cient of variation of R-R intervals also significantly decreased, but only after
exposure to 528 Hz music. Tension-
anxiety and Total Mood Disturbance
scores were significantly reduced after exposure to 528 Hz music, while there
was no significant difference following 440 Hz musi
c. These results suggest
that the influence of music on the autonomic nervous system and endocrine
system varies depending on the frequency of the music, and furthermore, that
528 Hz music has an especially strong stress-
reducing effect, even following
only five minutes of exposure.
Music, Stress, Salivary Assay, Autonomic Nervous System
It is well known that listening to music reduces stress. Research has investigated
How to cite this paper:
.L., Yamaguchi, T. and Kobayashi, H.
8) Effect of 528 Hz Music on the En-
docrine System and Autonomic Nervous
August 10, 2018
September 3, 2018
September 6, 2018
Copyright © 201
8 by authors and
Research Publishing Inc.
This work is licensed under the Creative
License (CC BY
K. Akimoto et al.
10.4236/health.2018.109088 1160 Health
many aspects of this phenomenon, and it is recognized that several constituent
elements of music are involved in this stress reduction effect. Previous studies
that have examined emotional arousal in response to listening to music have
suggested that music arouses different emotions depending on its characteristics,
such as melody, rhythm, and dynamism .
However, few studies have investigated how differences in the frequency of
music affect the human body. In a study in which rats were exposed to musical
stimuli of different frequencies, the rats’ blood pressure decreased depending on
the frequency; notably, this change was observed to a greater extent for music in
the frequency range of 16 kHz compared to 4 kHz frequency . This result
suggests that music including high-frequency sound stimulates dopamine syn-
thesis and suppresses sympathetic nervous system activity . Furthermore,
others report that listening to high-frequency music increases the activity of the
parasympathetic nervous system and reduces stress, in comparison to listening
to low-frequency music . Therefore, in the present study, we examined the
stress reduction effect of music with a frequency of 528 Hz by measuring its in-
fluence on the endocrine system and autonomic nervous system.
Music of the aforementioned frequency (528 Hz) has lately attracted attention
as “healing” music. Usually, the reference tone of tuning is 440 Hz, and this is
the international standard frequency (we refer to this as 440
). In this
musical scale, there is no 528 Hz note. However, setting the reference tone to 444
Hz means that 528 Hz is included in the musical scale. We refer to music that is
tuned and composed in this way as
528 Hz music
. In general, this specific type of
music using a scale including 528 Hz is called
ous effects have been ascribed to the solfeggio frequency, but none of these have
any scientific basis. Accordingly, we examined the effect of such music com-
pared to 440 Hz music.
In this study, we measured salivary biomarkers of stress (cortisol, chromogra-
nin A, and oxytocin), which can be collected noninvasively, as indices of stress
relief. It is known that listening to music affects the endocrine system and auto-
nomic nervous system.
In the endocrine system, cortisol and chromogranin A have been used as in-
dicators of stress. In a previous study, conducted in patients who had just un-
dergone the stressful experience of learning in detail about the procedures in-
volved in surgery that they would undergo the next day, salivary cortisol was
significantly reduced among a group who listened to music for one hour, com-
pared to a group who did not . Additionally, in another study, which admi-
nistered the Trier Social Stress Test (in which stress is caused to participants and
their salivary cortisol levels thereby raised), the increase of salivary cortisol in a
group who took the test while listening to relaxing music was significantly sup-
pressed compared to that of a control group .
Chromogranin A is present in the submaxillary duct and is released into saliva
by autonomic nerve stimulation . It has also been reported that, when psy-
chological stress is experienced, chromogranin A rises ahead of cortisol and de-
K. Akimoto et al.
10.4236/health.2018.109088 1161 Health
creases early after stress is relieved . Furthermore, others have examined the
effect on both cortisol and chromogranin A of listening to music  . Specif-
ically, they report that salivary cortisol and chromogranin A decrease signifi-
cantly before and after listening to music . In contrast, other researchers ob-
serve that, although salivary chromogranin A decreases significantly, there is no
significant difference in cortisol levels .
In addition to the above indicators, we also focused on oxytocin. The effect of
listening to music on oxytocin has recently attracted attention, with a previous
study indicating that salivary oxytocin increases when participants listen to
slow-tempo music compared to fast-tempo music .
As an additional objective index, we measured the activity of the autonomic
nervous system. Finally, we also administered the Japanese short version of the
Profile of Mood State, 2nd edition (POMS 2) to participants as a subjective index
of stress. Both of these measures have also been used in many previous studies
 . We consider these measures to support the data provided by the
above-mentioned salivary biomarkers of stress marker.
The 528 Hz music that we used in this study was soothing piano music. Some
researchers report that, depending on the mental state of the listener, music pre-
ferences vary . However, they also find that piano music is appreciated re-
gardless of the listener’s circumstances. Thus, we examined the stress-mitigating
effect of 528 Hz music from various perspectives. This study offers a new piece
of evidence for use in music therapy.
Participants were nine healthy adults, one man and eight women, aged 26 - 37
years (mean age: 31.1;
= 1.35). We recruited participants as volunteers re-
gardless their previous musical education. Everyone participated with full un-
derstanding of the study and gave their informed consent. They were asked to
abstain from caffeine and smoking for one hour before starting the experiment.
They did not apprise of which music they were listening to. This study was ap-
proved by the Ethics Committee of Shiba Palace Clinic, Tokyo, Japan (Protocol #
Figure 1 shows an outline of the experimental procedure. The study was carried
out in a conference room, where the room temperature was 25.0˚C - 25.5˚C and
humidity was 67% - 76%. In consideration of circadian variation in cortisol and
chromogranin A  , we started the experiment at 2 PM for each condi-
tion. Participants listened to the music from a seated position beside a speaker.
The music was soothing piano music, and the only difference between the two
musical conditions was in frequency, which was either 528 Hz or 440 Hz. Each
participant took part in each of the conditions on separate days.
K. Akimoto et al.
10.4236/health.2018.109088 1162 Health
Figure 1. The experimental procedure. Participants wore a Bio Information
Tracer (M-BIT) device and their electrocardiogram was recorded continuously
from the acclimation phase until 45 minutes after music exposure. Participants
listened to music for five minutes. Salivary samples were collected at five time
points: immediately before music exposure, immediately after music exposure, 15
minutes later, 30 minutes later, and 45 minutes later. The Profile of Mood State,
2nd edition (POMS 2) was administered before and after music exposure.
2.3.1. Salivary Assay
We collected salivary samples at five time points (immediately before listening to
music, immediately after listening to music, 15 minutes later, 30 minutes later,
and 45 minutes later) using the Saliva Collection Aid (Salimetrics LLC, USA).
We measured cortisol, chromogranin A, and oxytocin as salivary biomarkers of
stress. These were measured using the Salivary Cortisol Enzyme-Linked Immu-
nosorbent Assay (ELISA) Kit (Salimetrics LLC, USA), Human Chromogranin A
ELISA Kit (Yanaihara Institute Inc., Japan), and Oxytocin ELISA Kit (Arbor
Assays, USA), respectively.
2.3.2. Autonomic Nervous System
We recorded an electrocardiogram using the Bio Information Tracer (M-BIT)
(Institute of Man and Science Inc., Japan). Participants wore this small wearable
sensor (49 × 39 × 8 mm, 14 g) on the left side of the chest during an acclimation
phase, and data were recorded continuously until 45 minutes after music expo-
sure. The BIT analysis center analyzed these data in terms of low frequency (LF),
high frequency (HF), and the coefficient of variation of R-R intervals (CVRR),
and we received the averaged values of these measures over the five minutes
preceding each saliva collection time point.
2.3.3. Questionnaire on Mood States
Self-reported mood states were collected using the Japanese short version of the
POMS 2 scale before listening to music and 45 minutes after doing so. The
POMS 2 is a questionnaire developed in the United States as a questionnaire
method to evaluate mood. It is possible to quickly evaluate not only the state of
emotion that lasts for a relatively long time but also the state of temporary feel-
ings and emotions that change depending on the situation that subject is in. It is
K. Akimoto et al.
10.4236/health.2018.109088 1163 Health
used in a variety of fields such as clinical, workplace, and school. For example, it
has been applied to the course of treatment of mental disorders, mental changes
of people with physical disorders, screening in the workplace, and exercise or
The POMS 2 evaluates seven mood scales of anger-hostility (AH), confu-
sion-bewilderment (CB), depression-dejection (DD), fatigue-inertia (FI), ten-
sion-anxiety (TA), vigor-activity (VA), friendliness (F) at the same time. TMD is
a general indicator such as mood disorders, psychological distress, and subjec-
tive well-being. The TMD score is calculated by the sum of the elementary scores
of AH, CB, DD, FI, TA, and VA. (Since VA is weighted of negative, it is sub-
tracted from the sum of the other five). Therefore, the score is a comprehensive
evaluation of expression of negative mood.
The POMS 2 consists of 35 items questions and a respondent answer each of
question with the evaluation scale of POMS 2 (0 = not at all, 1 = slightly, 2 =
modestly, 3 = considerably, 4 = very). Although this evaluation scale is constant,
since the number of items differs depending on the scale, the degree indicated by
each scale score is not constant. In short, even though the absolute value of the
score is the same, the meaning of the score differs between scales. Since it is im-
possible to properly compare the scales at this score, the score is converted to a
standardized score, that is, a T score. The T score is a normalized standard of
assessment (average value is 50 and the standard deviation is 10) so that the
same value has equivalent meaning. The measurer interprets the score by con-
verting the graded evaluation scale into the T score.
Regarding the TMD score and negative mood state (AH, CB, DD, FI, and
TA), the higher the T score, the stronger the emotion concerning negative emo-
tion or mood disorder. Regarding the positive emotional state (VA, F), the high-
er the T score, the more positive emotion means .
2.4. Data Analysis
We used the Wilcoxon signed-rank test to detect any differences between time
points in each condition in this study. The threshold for statistical significance
was set at
3.1. Salivary Assay
In consideration of the response time lag of cortisol  , we took the mean
level of salivary cortisol measured immediately after music exposure as
representing the level immediately before music exposure, and so on; therefore,
data were only available until 30 minutes after music exposure, rather than 45
(Figure 2). After exposure to 528 Hz music, mean levels of salivary cortisol de-
creased with the passage of time and were significantly reduced 30 minutes later
(0.43 ± 0.04 → 0.25 ± 0.02,
< 0.011). In contrast, after listening to 440 Hz mu-
K. Akimoto et al.
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sic, mean levels of cortisol slightly decreased after 30 minutes, but there was no
significant difference between the time points.
3.1.2. Chromogranin A
There was no significant difference in chromogranin A levels following exposure
to either 528 Hz music or 440 Hz music (Figure 3). However, in the 528 Hz mu-
sic condition, mean levels of chromogranin A showed a tendency to fall until 30
minutes after music exposure. In contrast, in the 440 Hz music condition, mean
levels of chromogranin A showed a tendency to increase until 30 minutes later.
Figure 4 illustrates the effects of music on oxytocin. Mean levels of oxytocin in-
creased significantly immediately after listening to 528 Hz music (37.57 ± 1.53 →
73.58 ± 5.04,
< 0.038). After listening to 440 Hz music, mean levels of oxytocin
also increased, but the difference was not significant.
3.2. Autonomic Nervous System
An electrocardiogram could not be recorded correctly for one of the partici-
pants, so we analyzed the data from eight participants for this measure (Figure
5). In both conditions, the ratio of LF to HF decreased significantly immediately
after listening to music (528 Hz music: 77.89 ± 0.0025 → 62.34 ± 0.0032,
0.012, 440 Hz music: 72.52 ± 0.0028 → 54.72 ± 0.0037,
< 0.012), whereas CVRR
decreased significantly only immediately after listening to 528 Hz music (6.67 ±
0.00040 → 5.36 ± 0.00043
3.3. POMS 2
After exposure to 528 Hz music, all negative mood scores decreased. In particu-
lar, tension-anxiety (48.44 ± 2.28 → 43.67 ± 2.45,
< 0.0091) and Total Mood
Disturbance (45.56 ± 1.99 → 42.00 ± 2.00,
< 0.0487) decreased significantly.
Additionally, all positive mood scores tended to increase. In contrast, after ex-
posure to 440 Hz music, almost all scores showed a similar tendency to change
in the same direction as observed in the 528 Hz music condition, but there was
no significant difference for any score. Furthermore, anger-hostility scores (in-
dicating negative mood) slightly increased, and friendliness scores (indicating
positive mood) decreased (Table 1).
The purpose of this study was to examine the effect of 528 Hz music on the en-
docrine system and the autonomic nervous system using objective and subjective
measures. Based on salivary biomarkers, electrocardiogram, and a mood state
questionnaire, we found that stress levels were reduced following five minutes’
exposure to 528 Hz music, whereas this was not the case for 440 Hz music.
We found that mean levels of cortisol decreased and mean levels of oxytocin
increased after listening to 528 Hz music. In general, cortisol increases via the
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Table 1. Effects of music on mood as assessed by subscales of the Profile of Mood State,
Measures 528 Hz of Music 440 Hz of Music
Anger-Hostility 43.6 (1.75) 41.6 (1.53) 42.6 (2.00) 42.6 (2.40)
Confusion-Bewilderment 50.6 (3.10) 47.9 (2.43) 49.1 (2.63) 45.9 (2.77)
Depression-Dejection 47.2 (2.03) 44.4 (1.45) 46.4 (2.34) 45.9 (2.37)
Fatigue-Inertia 46.2 (3.04) 44.0 (2.56) 42.9 (2.42) 41.0 (3.06)
Tension-Anxiety 48.4 (2.28) 43.7 (2.45)** 44.3 (2.94) 42.4 (2.92)
Vigor-Activity 56.3 (2.11) 57.8 (4.11) 55.6 (3.53) 55.7 (4.48)
Friendliness 60.3 (3.40) 61.6 (4.22) 57.2 (4.22) 56.1 (5.20)
Total Mood Disturbance 45.6 (1.99) 42.0 (2.00)* 43.6 (1.99) 41.9 (2.48)
< 0.05; **
< 0.01 in pre-exposure vs post-exposure comparison using the Wilcoxon signed-rank test.
Figure 2. Effects of music on mean levels of salivary cortisol. Error bars represent
standard errors. Time points are as indicated in Figure 1 (Mean ± SE) *
Figure 3. Effects of music on mean levels of salivary chromogranin A. Error bars
represent standard errors. Time points are as indicated in Figure 1 (Mean ± SE).
K. Akimoto et al.
10.4236/health.2018.109088 1166 Health
Figure 4. Effects of music on salivary oxytocin. Error bars represent standard
errors. Time points are as indicated in Figure 1 (Mean ± SE) *
Figure 5. Effects of music on the autonomic nervous system, as measured by the ratio of
low frequency to high frequency (LF/HF) and coefficient of variation of R-R intervals
(CVRR). Time points are as indicated in Figure 1 (Mean ± SE) *
hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Oxytocin is
produced by the hypothalamus and released by the posterior pituitary gland. It is
well known that production of oxytocin increases in the process of breastfeeding.
In addition, some studies suggest that production of oxytocin increases in re-
sponse to pleasant sensations or positive social interactions  . When an
individual hears music, responses to these stimuli are eventually transferred to
the amygdaloid body via the medial geniculate body of the thalamus. Sensory
information that causes emotions, such as music, is processed in the amygdaloid
body circuit. Subsequently, signals are output to the hypothalamus, causing an
oxytocin response  . Furthermore, it is reported that oxytocin controls
activation of the HPA axis . Taking this information together with the
present findings, we infer that mean levels of oxytocin increased in our study
because the 528 Hz music was transmitted to the amygdaloid body as a pleasing
sensation. Additionally, the decrease in mean level of cortisol occurred as a re-
sult of the action of oxytocin. This suggests that participants felt comfortable lis-
tening to 528 Hz music and this stimulus reduced their stress level.
Mean levels of oxytocin also increased after listening to 440 Hz music, but no
significant difference was identified. In addition, mean levels of cortisol slightly
decreased in this condition after 45 minutes, but this difference also failed to
reach significance. In other words, we could not confirm that a stress mitigation
effect occurred in the 440 Hz music condition.
K. Akimoto et al.
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Mean levels of chromogranin A showed a tendency to decrease after listening
to 528 Hz music. Chromogranin A is released along with catecholamine via the
sympathetic-adrenal-medullary axis in response stress. Chromogranin A is found
in the duct of the submandibular gland. It is released into the saliva as a result of
stimulation of the autonomic nervous system   .
Concerning the autonomic nervous system, in both musical conditions, a sig-
nificant decrease in LF/HF was observed immediately after listening to music. LF
is considered to reflect the autonomic activity of both the sympathetic and the
parasympathetic nervous systems, whereas HF reflects parasympathetic activity,
and LF/HF reflects sympathetic activity  . Additionally, CVRR signifi-
cantly decreased immediately after listening to 528 Hz music. This measure
represents the coefficient of variance of the R-R interval (RRI) and is calculated
by dividing the standard deviation of the RRI by the mean of RRI. CVRR mainly
reflects regulation of parasympathetic nervous system activity . In other words,
a low value of this measure indicates concentration and a heightened sense of
tension . Taken together, the results of the Chromogranin A, LF/HF, and
CVRR analyses suggest that when the participants listened to 528 Hz music, they
concentrated on listening to the music while also relaxing.
Interestingly, after listening to 440 Hz music, mean levels of chromogranin A
showed a tendency to increase, while LF/HF significantly decreased in both con-
ditions. At the same time, the cortisol, oxytocin, and POMS 2 measures did not
show stress reduction. This might suggest that people were relaxed by listening
to music but did not feel comfortable with 440 Hz music.
The results of the POMS 2 showed that after listening to 528 Hz music, ten-
sion-anxiety and Total Mood Disturbance scores decreased significantly. After
listening to 440 Hz music, almost all scores showed a tendency to change in a
similar way as in the condition with 528 Hz music; however, not only was there
no significant difference in any of the scores, but anger-hostility slightly in-
creased and friendliness decreased. On the basis of these results, it can be con-
cluded that participants also felt subjectively relaxed after listening to 528 Hz
Overall, we found that participants experienced objective and subjective stress
reduction after listening to 528 Hz music, while this effect could not be con-
firmed for 440 Hz music. In this study, participants listened to music only for
five minutes in each condition. In most previous studies, participants have lis-
tened to music for 10 minutes or longer. Therefore, it might be the case that lis-
tening to 440 Hz music for longer would evoke the stress-reducing effect, espe-
cially as indexed by chromogranin A and the POMS 2, as in previous studies.
However, the point remains that even if the exposure to music lasts only five
minutes, listening to 528 Hz music reduces stress to the endocrine system and
the autonomic nervous system. Although the sample size of this study was li-
mited, these are interesting data and could provide support for a new approach
in music therapy.
K. Akimoto et al.
10.4236/health.2018.109088 1168 Health
We found that music of different frequencies had different effects on the endo-
crine system, especially oxytocin and cortisol. Our findings suggest that 528 Hz
music reduces stress even if participants listen to the music for only a short time.
We thank the participants for volunteering and the Institute of Man and Science
Inc. for analyzing the electrocardiogram data.
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