Open Journal of Urology, 2013, 3, 173-178
http://dx.doi.org/10.4236/oju.2013.34032 Published Online August 2013 (http://www.scirp.org/journal/oju)
Testosterone Levels Do Not Decline with Age
in Healthy Men
Akanksha Mehta1*, Alexander Bolyakov1, Raymond C. Sultan1, Laurent Vaucher2,
Anna Mielnik1, Joseph Kiper1, Darius A. Paduch1
1Department of Urology, Weill Cornell Medical College, New York, USA
2Department of Urology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
Received June 3, 2013; revised July 1, 2013; accepted July 9, 2013
Copyright © 2013 Akanksha Mehta et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Aim: To establish norms for reproductive hormones in healthy males with normal urologic and sexual function, and to
assess the change in reproductive hormone levels with respect to age among healthy males. Methods: Healthy volun-
teers aged 18 - 29 (group 1) and 45 - 65 (group 2) were recruited for enrollment in a prospective study. Inclusion criteria
comprised normal urinary, ejaculatory, orgasmic, and erectile function, as determined by IPSS, MSHQ, and IEFF-15.
Men with history of chronic medical illnesses or chronic medication use were excluded. Fifty men met the study criteria.
Each participant underwent physical examination and standardized serum hormone evaluation using ELISA and EIA
methods. Statistical analysis was performed using JMP 8.0 software (SAS Institute Inc., Cary, NC), to compare hor-
mone levels between the two groups of men. Results: There were 25 men each in groups 1 (mean age 26) and 2 (mean
age 51). Overall, 46% were Caucasian, 31% African American, 15% Hispanic, and 8% Asian. There was no statistically
significant difference in serum total T, SHBG, E2, or LH between groups 1 and 2. Notably, the 95% CI for serum T
levels for both groups of men were considerably higher than commonly accepted lower-end cut-off limits. Conclusion:
Serum T levels do not vary significantly with age, in otherwise healthy men with normal sexual and urologic function.
Furthermore, healthy men have much higher serum total and free T levels than the lower-end reference limits provided
by commercial laboratories.
Keywords: Testosterone; Aging; Hypogonadism; Reference Values
1. Introduction
Hypogonadism is a clinical condition characterized by
low levels of serum testosterone (T), in association with
specific signs and symptoms of low T, which may in-
clude physical, sexual, reproductive, and cognitive ef-
fects. Hypogonadism affects an estimated two to four
million men in the United States; its prevalence increases
with age [1]. When hypogonadism occurs in older men,
the condition is often called andropause, or androgen
deficiency of the aging male [2].
Testosterone supplementation for the treatment of hy-
pogonadism has increased substantially in the United
States over the past few years, with an increase of more
than 500% in prescription sales of testosterone products
since 1993 [3]. Testosterone replacement therapy (TRT)
results in a demonstrable improvement in symptoms of
hypogonadism, including diminished libido, energy and
well-being, impaired cognition, decreased muscle mass/
strength, anemia, osteoporosis, erectile dysfunction, and
visceral obesity [4-13].
Because the diagnosis of hypogonadism requires labo-
ratory confirmation of a low level of testosterone, clini-
cians rely heavily on laboratory reference ranges to de-
termine whether a patient may be a candidate for TRT.
However, at present there is no universally accepted
definition of hypogonadism, no universally used assay
for the analysis of serum T, and no universally ag-
reed-upon indications for the initiation and use of TRT.
Guidelines from the Endocrine Society maintain that T
levels below 300 ng/dL are diagnostic of hypogonadism,
while higher levels are normal [14]. Accordingly, the
Society only recommends TRT in men with unequivo-
cally low testosterone concentrations and symptoms as-
sociated with androgen deficiency [14]. Meanwhile, a
consensus statement from the ISA, ISSAM, EAU, EAA,
and ASA recommends that T levels above 350 ng/dL do
*Corresponding author.
C
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A. MEHTA ET AL. 174
not require treatment, while levels below 230 ng/dL do
[15]. Yet other authorities emphasize the importance of
the symptoms of androgen deficiency over serum bio-
chemistry, suggesting that men may be “hypogonadal” at
the tissue level while having a normal T level [16,17].
The confusion stems, in part, from the variability in
testosterone assays and their interpretation. Historically,
the reference range for serum testosterone reported by
scientific publications was based on traditional radio-im-
munoassay (RIA) methods, and ranged from 300 - 1000
ng/dl (10.4 - 34.7 nmol/liter) [18]. Over the last decade,
techniques for testosterone measurement have evolved
from RIA, to automated enzyme-linked immunoassays
(EIA) available in most laboratories, and liquid chroma-
tography-mass spectroscopy methods in reference labo-
ratories. As a result, widely variable reference ranges
have been reported for different assays, spanning low-
end clinical detection limits of 170 - 200 ng/dL to up-
per-end limits of 700 - 800 ng/dL [18]. However, these
reference ranges are not specific for healthy men with
normal sexual and reproductive function, and are rarely
adequately adjusted for age [19,20]. Consequently, an
objective comparison of results from different published
studies, using different assays, is fraught with error.
The objective of this study was two-fold. Firstly, we
aimed to establish norms for reproductive hormones
based on a diverse population of healthy males with nor-
mal urologic and sexual function. Secondly, we aimed to
assess the change in reproductive hormone levels with
respect to age, in a cross sectional analysis of healthy
men from two different age groups.
2. Materials and Methods
2.1. Study Population
Two groups of healthy male volunteers were recruited
for enrollment into this prospective study via informa-
tional study flyers distributed amongst New York Pres-
byterian Hospital-affiliated medical facilities, and adver-
tisements in local newspapers. Group 1, the younger
group, consisted of men between the ages of 18 - 29
years, while group 2, the older group, consisted of men
aged 45 - 65 years. All individuals who expressed inter-
est in the study were pre-screened by the study coordi-
nator (JK). Individuals with any history of a chronic
medical condition or chronic medication use were ex-
cluded. Others were invited for enrollment on a first-co-
me first-served basis.
Following pre-screening, all eligible volunteers comp-
leted the International Prostate Symptom Score (IPSS),
International Index of Erectile Dysfunction (IIEF-15),
and Male Sexual Health Questionnaire (MSHQ). All
volunteers also underwent a complete physical examina-
tion by an attending physician (DAP). Only men with a
normal physical exam, and normal erectile, ejaculatory,
orgasmic, and voiding function, were included in the
study. A total of 50 men were enrolled into the study,
divided equally between groups 1 and 2. All study par-
ticipants were provided a modest inconvenience fee.
Approval for this study was provided by the Weill
Cornell Medical College Institutional Review Board.
2.2. Measurement of Hormone Levels
All subjects were scheduled for baseline hormone eva-
luation between the hours of 7 and 10 a.m. A peripheral
blood sample was obtained from each study participant
(25 mL), centrifuged for 30 minutes, and the serum
transferred to a collection tube. All samples were main-
tained at −20˚C prior to completion of hormonal assays,
following which samples were transferred to −80˚C. The
hormones measured included: total testosterone (tT), free
testosterone (fT), sex-hormone binding globulin (SHBG),
estradiol (E2), follicle stimulating hormone (FSH), lu-
teinizing hormone (LH), dehydroepiandrosterone (DHEA)
and dehydroepiandrosterone sulfate (DHEA-S). All
hormones were measured in duplicate, on the day of col-
lection, by the same technician (AB), using commercially
available enzyme-linked immunosorbent assays (ELISA)
and enzyme immunoassays (EIA).
For each assay, micro plates were read using Spectra-
Max 340 PC micro-plate spectrophotometer, controlled
by Soft Max Pro data analysis software (Molecular De-
vices, Sunnyvale, CA), and concentrations calculated
using four-parameter curve fit. Absorbance was meas-
ured at 450 nm, and dual wavelength correction at 620
nm was used in all assays. Standards and controls pro-
vided by manufacturer were included with each run and
on each plate.
Biorad Lyphochek Assayed Chemistry Control Bilevel
packs (Bio-Rad Laboratories, Hercules, CA) were used
for quality control of assays. All measures were per-
formed in duplicates. Samples with results exceeding
dynamic range were diluted using charcoal stripped ster-
oids free serum, and re-analyzed. In order to calculate
inter-assay variability, ten randomly-selected subjects
from each group underwent repeat hormone evaluation
per the study protocol, within 6 months of the initiation
of the study.
2.3. Statistical Analysis
All patients in group 1 completed the study. Three pa-
tients from group 2, aged 56, 57, and 59 years, were un-
able to complete the study, and were excluded from the
final analysis. All results were entered in a study specific
database and analyzed using JMP 8.0 statistical software
(SAS Institute Inc., Cary, NC), to compare the various
hormone levels between the two groups of men using

A. MEHTA ET AL. 175
Student’s t-test. A p-value < 0.05 was considered statisti-
cally significant. GraphPad Prism 5.0 (GraphPad Soft-
ware Inc., La Jolla, CA) was used to test for normal dis-
tribution of hormonal results and to verify the calculated
hormone concentrations obtained from SoftMax Pro
(Molecular Devices, Sunnyvale, CA).
3. Results
Twenty-five men each were enrolled into group 1 (mean
age: 26) and group 2 (mean age 51). Demographic data
was available for 48 subjects. Overall, 46% (22/48) of
men were Caucasian, 31% (15/48) African American,
15% (7/48) Hispanic, and 8% (4/48) Asian. Ninety-five
percent of study subjects identified themselves as being
heterosexual.
Details of the manufacturers, catalog numbers, re-
ported reference ranges, minimum and maximum limits
of detection, and inter-assay variability for each of the
hormone assays used in this study is summarized in Ta-
ble 1. The laboratory co-efficient of variance was ≤6.2%
for each assay, indicating good reproducibility with lim-
ited inter-assay variability.
Serum hormone levels of tT, fT, SHBG, E2, FSH, and
LH, DHEA, and DHEA-S in the two groups of men are
listed in Table 2. There was no statistically significant
difference noted in serum tT (766 vs. 599 ng/dL), SHBG
(25.1 vs. 32.5 nmol/L), E2 (17.1 vs. 22.7 pg/mL), or LH
(5.37 vs. 7.56 mIU/mL) between groups 1 and 2, respec-
tively. Men in group 1 had higher levels of serum fT
(74.5 vs. 50.9 pg/mL, p = 0.004), DHEA (11.4 vs. 7.58
ng/mL, p = 0.04), and DHEA-S (2.84 vs. 1.35 ug/mL, p
< 0.001), compared to the older cohort of men in group 2.
Serum FSH levels were lower in group 1 compared to
group 2 (1.97 vs. 5.00 mIU/L, p < 0.001).
Compared to manufacturer-provided reference ranges
(Table 1), the 95% CI values for serum tT, fT, DHEA,
and DHEA-S were higher among the study subjects in
both groups 1 and 2 (Table 2). Interestingly, the 95% CI
range for serum FSH was both lower and tighter among
the study subjects, who represent healthy men, than the
reference ranges that have traditionally been reported by
manufacturers.
4. Discussion
Our results demonstrate that serum testosterone levels are
not significantly different in healthy men between the
second and sixth decades of life. Furthermore, the range
of distribution of serum testosterone levels in the study
subjects was considerably higher than what is commonly
reported by manufactures of commercially available tes-
tosterone assays, indicating that reference ranges for
healthy men with normal sexual and urologic function
may be different than the references ranges for the gen-
eral population of men. These findings are in contrast to
recent reports from longitudinal studies of aging men,
which show that serum testosterone levels decline with
each decade of life [21-23]. However, these popula-
tion-based studies included men over the age of 65, and
did not specifically select for healthy men without any
medical comorbidities.
To our knowledge, to date, serum reference ranges of
reproductive hormones have not been studied in a diverse
population of healthy men with documented normal uro-
logic and sexual function. The evaluation of orgasmic
and ejaculatory function in healthy men with normal
sexual function is difficult because of the ethical issues
involved, concerns for privacy and confidentiality, in-
creased scrutiny by internal review boards towards stud-
ies of human sexuality, and complex logistical issues
involved in screening and enrolling patients for such
studies. Furthermore, studies of healthy subjects are ex-
pensive, and difficult to obtain funding for, as no clinical
benefit of “pharmacological” treatment can be proven in
studies of normal subjects. Nevertheless, the importance
of such studies, to establish a baseline reference range,
cannot be emphasized enough.
Assays for the measurement of serum testosterone
have been criticized for their unreliability [24,25], and
variable limits of detection. Taieb et al. compared testos-
terone levels for 116 samples (50 males, 55 females and
11 children) measured by 10 different commercial kits
with results obtained by isotope dilution-mass spectro-
metry, and concluded that none of the immunoassays
tested was sufficiently reliable for the investigation of
sera from children and women, in whom very low testos-
terone concentrations are expected [26]. There are sig-
nificant intra-individual fluctuations in serum testoster-
one levels, and a wide range of testosterone levels in the
general male population. Thus, it is likely that the vari-
ability between assays derives in large part from failure
to establish specific norms for a healthy, young to mid-
dle-aged population of men. Additionally, many manu-
facturer-reported references ranges assume the serum
testosterone follows a normal distribution, which is not,
in fact, the case. As a result, manufacturer-quoted refer-
ence ranges are often too low, making them less sensitive
in detecting hypogonadism [27]. This assertion is cer-
tainly supported by our data, which demonstrates a
higher range of distribution of serum testosterone levels
in the study subjects, compared to published reference
ranges (Table 1).
While several cross sectional studies have demon-
strated lower concentrations of circulating total and/or
free T in older men [28-37], other have shown that T lev-
els do not fall significantly with age in exceptionally
healthy men [38,39], questioning the relative roles of
age-related illness versus aging per se in producing the

A. MEHTA ET AL. 176
Table 1. Manufacturer reported reference ranges, limits of detection, and laboratory co-efficient of variance for the assays
used.
Hormone Type of Assay Manufacturer Catalog No. Manufacturer Reported
Reference Range Limits of Detection Lab Co-efficient of Variance (%)
T EIA Beckman Coulter;
Webster, TX DSL-10-4000 290 - 990 ng/dL (20 - 30 yr old); 160 - 660 ng/dL (50 - 60 yr old) 100 - 2500 ng/dL 6.2
Free T EIA Beckman Coulter;
Webster, TX DSL-10-491006.2 - 28.1 pg/mL 0.25 - 100 pg/mL 1.9
LH ELISA Beckman Coulter;
Webster, TX DSL-10-4600 1.08 - 8.34 mIU/mL 1 - 100 mIU/mL 3.5
FSH EIA ALPCO; Salem, NH 11-FSHHU-E011.0 - 18.0 mIU/mL 5 - 100 mIU/mL 2.8
SHBG EIA ALPCO; Salem, NH 11-SHBHU-E017.0 - 70.0 nmol/L 3.3 - 295 nmol/L 5.1
E2 EIA ALPCO; Salem, NH 20-DR-4399 10.0 - 36.0 pg/mL 3 - 200 pg/mL 2.8
DHEA EIA Beckman Coulter;
Webster, TX DSL-10-9000 1.8 - 12.5 ng/mL 0.2 - 27 ng/mL 1.7
DHEA-S EIA ALPCO; Salem, NH 11-DHEHU-E010.39 - 4.63 ug/mL 0.005 - 10 ug/mL 2.9
EIA = Enzyme immunoassay; ELISA = Enzyme-linked immunosorbent assay; T = Testosterone; LH = Luteinizing hormone; FSH = Follicle stimulating hor-
mone; SHBG = Sex-hormone binding globulin; E2 = Estradiol; DHEA = Dehydroepiandrosterone; DHEA-S = Dehydroepiandrosterone sulfate.
Table 2. Means and distribution of serum hormone levels (reported as 95% CI) in healthy men of different age groups.
Group 1 (Age 18 - 29) Group 2 (Age 45 - 65) Statistical significance
Total T (95% CI) 766 (569 - 963) ng/dL 599 (497 - 700) ng/dL p = 0.11
Free T (95% CI) 74.5 (60.6 - 88.4) pg/mL 50.9 (44.2 - 57.6) pg/mL p = 0.004
SHBG (95% CI) 25.1 (15.8 - 34.4) nmol/L 32.5 (26.4–38.7) nmol/L p = 0.16
E2 (95% CI) 17.1 (11.4 - 22.7) pg/mL 22.7 (9.67 - 35.8) pg/mL p = 0.42
FSH (95% CI) 1.97 (1.32 - 2.61) mIU/mL 5.00 (3.67 - 6.34) mIU/mL p < 0.001
LH (95% CI) 5.37 (2.62 - 8.13) mIU/mL 7.56 (3.42 - 11.7) mIU/mL p = 0.36
DHEA (95% CI) 11.4 (7.97 - 14.8) ng/mL 7.58 (6.15 - 9.02) ng/mL p = 0.04
DHEA-S (95% CI) 2.84 (2.26 - 3.42) ug/mL 1.35 (1.04 - 6.66) ug/mL p < 0.001
observed decreases in serum testosterone. In the present
study, which strictly excluded study subjects on the basis
of any history of chronic medical illness or medication
use, no significant decrease in serum testosterone was
noted with increasing age. The utility of age-adjusted
norms for serum testosterone levels has long been de-
bated in the literature [27,40,41]. However, the present
data suggests that establishing norms based on healthy
men may be more important and utile than using age
adjusted norms.
Lazarou et al. [19] point out that if hypogonadism af-
fects 15% - 35% for men over the age of 50 years [1], but
only 2.5% of values are categorized as “low,” then a
large majority of affected men will fail to be correctly
identified as hypogonadal. Men who stand to benefit
from TRT may not be offered treatment. The use of
age-adjusted values may decrease the test sensitivity for
detecting androgen deficiency in aging males [42]. While
the use of age-adjusted norms may make sense for the
statistical representation of a population, it has no clinical
justification for the diagnosis of hypogonadism [19]. Our
data lends supports the possibility that the use of norms
based on healthy men with normal sexual function may
optimize clinical care of both older and younger men.
One limitation of this study is the small sample size of
healthy volunteers recruited for participation. Validation
of our results in a larger and equally diverse population
of healthy men would be beneficial. Secondly, the en-
rollment criteria did not specifically require documented
paternity or other evidence of normal reproductive func-
tion, such as semen analyses. While normal reproductive
function was assumed among the study subjects, based
on a normal urologic, medical, and sexual history, con-
firmation of paternity or normal semen analysis would
further strengthen the findings of this study. The gold
standard assay for the measurement of serum testosterone
is liquid chromatography-mass spectroscopy, used pri-
marily in reference and research laboratories [43]. Nev-
ertheless, for this study, we chose to use EIA and ELISA
assays for the measurement of reproductive hormones in
our study population, because these methods are com-
mercially available and, therefore, more widely used in
investigative laboratories.
In summary, healthy men with normal sexual function
have much higher total and free testosterone levels, than
the “normal” reference ranges commonly reported by

A. MEHTA ET AL. 177
commercial assays. In healthy men, with normal urologic
and sexual function, serum testosterone levels do not
appear to vary significantly with age. These findings are
important for the diagnosis and treatment of hypo-
gonadism in the general male population.
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