Materials and Methods

The Mean Testosterone Level is Higher in Adult Males With Deeper Voice
Ben Hoffman, Sid Mirgati, Tim Turnbaugh
Department of Biological Science
Saddleback College
Mission Viejo, CA 92692
Testosterone is an anabolic steroid hormone in males that has variety of responsibilities
including fetal development of genitalia and physical maturation during puberty; thus it
can be hypothesized that testosterone levels are directly related to the deepness of voice in
human males. To test this, a Salimetrics (®) kit was used to measure the amount of free
testosterone in subjects’ saliva. A micro-plate reader was used to provide a quantified
testosterone concentration reading. The voice and saliva of ten (n=10) adult males was
collected. Testosterone levels fluctuate throughout the day, thus multiple samples were
collected and the mean value of the samples was used in calculations. One set of the lowest
note a subject could produce was also collected. After the analysis, the mean voice
frequency of all subjects was measured and the samples were divided into two groups. The
first group included the subjects with lower voice frequency than the total average
frequency. The second group included the subjects with higher voice frequency than the
total average frequency. The mean testosterone level of first group was 626.139 pg/mL
(S.E.M ±134.728 pg/mL, C.L. ±428.766). The mean testosterone level of second group was
475.896 pg/mL (S.E.M ±35.546 pg/mL, C.L. ±91.375). To compare the mean testosterone
value of each group, a one-tailed unpaired t-test was performed (P=0.179). The results
rejected the hypothesis and stated there is no significant correlation between the
testosterone level and voice frequency in adult males.
Introduction (Center all headings)
Testosterone - a key hormone
present in every man - is responsible for
many functions and developments in the
human male. It is responsible for fetal
development of the male genitalia, the
physical changes that occur during male
puberty, and a variety of other functions in
the adult male, including sperm production,
erections, sex drive, muscle tone, and bone
health (Walker, et al.,1997).
In men, 95% of testosterone is
created in the testes and the rest is produced
in the adrenal glands. There are two major
forms of testosterone in human body: bound
testosterone and free testosterone. Bound
testosterone is attached to another substance
such as globulin or albumin. When the
testosterone is attached to another molecule
(what other molecule?), it is modified and
subsequently cannot fit into a receptor site.
Since it cannot become active, it is
eventually excreted by the body. Free
testosterone is the amount of testosterone
that floats through the blood on its own
(verbiage) and is readily available for use by
the body. In men, 35 to 155 pg/mL
(picograms per milliliter) is the normal
range of free testosterone. There may be
daily variations as male testosterone
levels peak in the morning and then steadily
decline until late evening. Stress, lack of
sleep and many other factors can affect
testosterone levels (Meuser, 1977).
When getting the testosterone levels
tested, the results may be misleading. If the
level of bound testosterone is within normal
range, it is not necessarily an indication of
healthy levels. The level of free testosterone
is more important, because free testosterone
is the one that is more readily used by the
body (Dabbs, Mallinger, 1999).
Taking steroid hormones such as
testosterone for performance enhancement
in males can cause testicles to shrink and
breast tissue to growth. For women, it can
cause a deepened voice, an enlarged clitoris,
hair loss from the head, and hair growth on
the body and face. In both genders, steroid
abuse can cause acne, mood swings,
aggression, and other problems. A simple
error in diet, such as eating a lot of sugar or
high-glycemic foods, restricting fat, being
abstaining from meat, or not getting enough
magnesium, zinc, or vitamin D may lower
testosterone levels. These lowered
testosterone levels may affect the functions
of individual development, immune system,
libido, heart health, energy, weight control
and emotional well-being (Apicella,
Feinberg 2009).
In men, the different depths of voice
are influenced by different concentrations of
circulating sex hormones and also by the
androgen sensitivity of the target organs
(Meuser, 1977). According to a Georgia
State University experiment, salivary
testosterone levels were “significantly
associated with lower pitched voices among
males but not among females (Dabbs,
1999).” Harries performed a similar study
looking at how testosterone level affects the
males voice during puberty. Their study
concluded that testosterone was not
“predictive of the changes, (but) there was a
correlation with testis volume and
testosterone levels." Alternative test
methods, including multiple saliva samples
from the Human Cognitive Neuroscience
Unit of the School of Psychology and Sport
Sciences, indicated even greater magnitude
of the relationship between high testosterone
and lower frequency. Some have
hypothesized lower voices have had an
evolutionary benefit of attracting females by
demonstrating a high level of testosterone.
This has been debunked, however, as a
Harvard study demonstrated that while men
prefer higher pitched women, women
express no preference for the reverse on
males (Apicella, 2009).
Our study tests the correlation
between the testosterone levels and the
depth of voice in men. Since the
testosterone fluctuates considerably from
one time to the next we decided to collect
multiple saliva samples and use the average
of testosterone levels for the calculations.
Based on past results, there is testosterone in
saliva, and the average levels detected were
295 ± 36 pg/ml in adult males (Dabbs,
Mallinger, 1999).
Our researchers hypothesize that the
mean testosterone level is going to be higher
in men with lower voice frequency.
Materials and Methods
Ten adult subjects were used in this
study. Subjects were adult male students at
Saddleback College, Mission Viejo,
California. All measurements were made
on the 20th of November 2013. Subjects
were contacted before and asked to
participate in the study. Each participant
signed an experiment specific agreement
waiver and filled out a short questionnaire.
After (an) agreement was reached, we
scheduled the day we would collect the
saliva and voice samples from the subjects.
On the scheduled day (November 20,
2013) the subjects were all present for the
first set of sample-collecting between 8:20
to 8:40 am. Each subject was given a saliva
collection aid and a pre-labeled collection
vial. Subjects were asked to insert the
ribbed-end of the saliva collection aid in the
pre-labeled collection vial and allow saliva
to pool in mouth before forcing the saliva
through the saliva collection aid into the vial
and fill approximately half the vial (1 mL).
Then the saliva collection aid was removed
and discarded and the cap was attached to
the collection vial. It was made sure that the
subjects passively drooled into their labeled
collection container. Directly after saliva
retrieval the researchers placed the test
containers into a cold storage filled with ice
to freeze the samples(;) preventing
degradation (excessively wordy). All the
voice sample-collecting (sample collection)
occurred (took place) outside room SM 244
at Saddleback College, Mission Viejo,
California. Subjects were asked to make
(produce) the lowest note they were able to
produce for 3 seconds. This was recorded
using an iPhone 4S which yielded a
collection rate of 44600 samples per second.
The sample-collecting occurred when no
other individual (except the subject himself
and the researcher that was holding the
recording device) was in (within)
approximately 5 yards of the subject to
minimize the background noises in the
recordings as much as possible. The second
set of samples was collected under identical
circumstances between 11:20 and 11:40 am.
The second set of saliva samples was also
placed into a cold storage filled with ice to
freeze the samples preventing degradation.
During the sample-collecting, each sample
was labeled as a number in order to keep
(retain) subject's privacy. In (delete ‘In’)
between collection and analysis the samples
were stored in a freezer at -4 degrees
The analysis of testosterone samples
occurred on November 22, 2013 in room
SM 244 at Saddleback College, Mission
Viejo, California. The researchers brought
out all of the sample containers from the
freezer at 8 am so the samples would be
thawed in time for analysis. The reagents
were also removed from the refrigerator the
day before in order to come (bring up) to
room temperature. The Salimetrics (®)
preparation and analysis procedure was
followed providing us with our raw data.
Voice recordings were analyzed
using Speech Analysis Software (SIL
International, Dallas, Texas) to find the
fundamental frequency (average pitch of the
sample) of the subject's voice. The equation
used by the Speech Analysis Software is a
modified version of Cepstrum analysis
𝐹𝑓𝑢𝑛𝑑𝑎𝑚𝑒𝑛𝑡𝑎𝑙 = 𝐶(𝑥) =
|ℱ −1 {log⁡(|ℱ{𝑓(𝑡)}|2 }|2 where ℱ represents
a fourier (Fourier) transformation of the
equation. The results of this equation
changed the wave form into a more linear
function making a calculation of average
frequency possible within the Speech
analysis software.
The data was run through a
Microsoft (®) Office excel (Excel)
spreadsheet. The program took the input
data obtained from the microreader and
outputted a 4- Parameter Sigmoid Minus
Curve. The curve was adjusted to fit along
the plot points of the standards as best as
possible (minimum asymptote: 0.1,
steepness -1.2, inflection point 100, and
maximum asymptote 1). This curve allows
the calculation of the testosterone
concentration in the unknown saliva samples
as demonstrated in Figure 1.
(indent) For the first group (subject with
lower voice frequency than the total
average) the mean testosterone level was
626.139 pg/mL (S.E.M ±134.728 pg/mL,
C.L. ±428.766). For the second group
(subject with higher voice frequency than
the total average) the mean testosterone
level was 475.896 pg/mL (S.E.M ±35.546
pg/mL, C.L. ±91.375). These data are
shown in Figure Two.
Ratio of optical density (B/Bo)
Log of Concentration (pg/ml)
Figure 1. Graph relating the Percent Bound of
Standards and Unknowns with the Log of
Testosterone Concentration (n=10). As the log of
concentration increases, the percent bound for
standards and unknowns decreases. The minimum
asymptote was 0.1, the steepness was -1.2, the
inflection point was 100, and the maximum
asymptote was 1.
(indent) After the testosterone
concentrations for each well was (were)
gathered, they were arranged according to
test and subject. The mean value of
testosterone levels for each subject was
calculated using:
𝑖=1 𝑡𝑖
= 𝑀𝑒𝑎𝑛⁡𝑇𝑒𝑠𝑡𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒
The mean value of the voice frequency level
from all subjects (n=10) was calculated 87.2
(indent) The subjects were separated into
two groups: the first group includes the
subjects with lower voice frequency than the
total mean. The second group includes the
subjects with the higher voice frequency
than the total mean. For each group, the
mean of testosterone level and voice
frequency was obtained and analyzed
Mean of patients' free
testosterone level (pg/mL)
Below Total Average Above Total
Figure 2. Mean Testosterone Concentration level
compare to the Mean Frequency of Voice in each
subject (n=10). The average voice frequency was
measure 87.2 Hz. There was no significant difference
in the mean testosterone level of subjects with voice
frequency of below total average in compare to the
subjects with higher voice frequency of the total
average. (p=0.179, one-tailed unpaired t-test). Error
Bars are mean ± SEM.
(indent) Based on the p value (P=0.179), we
can reject our hypothesis and accept the null
hypothesis that there is no significant
difference in the mean testosterone level in
relation to fundamental voice frequency.
(indent) Two other studies also concluded
testosterone levels have no significant
relationship to voice frequency in men
(Hughes et .l, 1997) (Meuser and Nieschlaq,
1977). In our experiment the testosterone
levels in subjects were significantly higher
than the normal adult males. The possible
explanation for this difference is the
performance errors during the saliva
preparation that could cause the incorrect
ratio of the sample to reagent. This error
could lower the number of reactions
between the antibodies and the testosterone
in Salimetric (®) plates and eventually
demonstrate higher testosterone level than
the actual level.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA
Author (s): Ben Hoffman, Sid Mirgati, Tim
Apicella, Coren and David Feinberg. 2009.
Voice Pitch Alters Mate-Choice-Relevant
Perception in Hunter-Gatherers. Proceeding
of the Royal Society. Vol. 276:1077-1082.
Dabbs, James and Alison Mallinger. 1999.
High Testosterone Levels Predict Low
Voice Pitch Among Men. Science Direct.
Vol. 27, Issue 4.
Evans, Sarah, Nick Neave, Delia Wakelin,
and Colin Hamilton. 2008. The
Relationship Between Testosterone and
Vocal Frequencies in Human Males.
Science Direct. Vol. 93, Issue 4-5.
Hughes, J M Walker, D M Williams, and S
Hawkins. 1997. Changes in the Male Voice
at Puberty. Archives of Disease in Children.
Meuser W. Nieschlaq. 1977. Sex hormones
and depth of voice in the male (Article in
Germany). Deutsch Med Wochenschr.
Title: The Mean Testosterone Level is
Higher in Adult Males With Deeper Voice
Summarize the paper succinctly and dispassionately. Do
not criticize here, just show that you understood the paper.
Testosterone affects many body functions
and features. One of them is the voice. The
average frequency of the male voice was
compared to the observed testosterone level.
No significant correlation was made
between the two.
General Comments
Generally explain the paper’s strengths and
weaknesses and whether they are serious, or
important to our current state of knowledge.
It was interesting to find the testosterone did
not significantly play a role in the depth of
the male voice. Perhaps this knowledge can
be extended to future male studies where
testosterone’s limits as a steroid are
The methods section was a bit wordy and
can use some work.
Good discussion and follow up on the
Technical Criticism
Review technical issues, organization and clarity.
Provide a table of typographical errors, grammatical
errors, and minor textual problems. It's not the
reviewer's job to copy Edit the paper, mark the
 This paper was a final version
 This paper was a rough draft
The authors forgot to create a discussion
section and instead put their discussion in
their results section.
There were few grammatical/spelling errors
throughout though word choice needs to be
looked over.
Introduction is logically organized.
 This paper should be published as is
 This paper should be published with
 This paper should not be published
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