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
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,
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 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 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. All the voice samplecollecting occurred outside room SM 244 at
Saddleback College, Mission Viejo,
California. Subjects were asked to make 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
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
subject's privacy. In between collection and
analysis the samples were stored in a freezer
at -4 degrees Celsius.
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 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 represents
a 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.
Results
The data was run through a
Microsoft Office excel spreadsheet. The
program took the input data obtained from
the microreader and outputted a 4Parameter 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.
Ratio of optical density (B/Bo)
1.2
1
0.8
0.6
0.4
0.2
0
1
10
100
1000
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.
After the testosterone concentrations for
each well was gathered, they were arranged
according to test and subject. The mean
value of testosterone levels for each subject
was calculated using:
∑𝑁
𝑖=1 𝑡𝑖
2𝑁
= 𝑀𝑒𝑎𝑛⁡𝑇𝑒𝑠𝑡𝑜𝑠𝑡𝑒𝑟𝑜𝑛𝑒
The mean value of the voice frequency level
from all subjects (n=10) was calculated 87.2
Hz.
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
separately.
For the first group (subject with lower voice
frequency than the total average) the mean
Mean of patients' free
testosterone level (pg/mL)
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.
800
600
400
200
0
Below Total Average Above Total
Average…
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.
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.
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.
Citations
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.
77:445-447.
Meuser W. Nieschlaq. 1977. Sex hormones
and depth of voice in the male (Article in
Germany). Deutsch Med Wochenschr.
25:102(8):261-4.