Caffeine intake is not associated with serum testosterone levels in adult men – cross-sectional findings from the NHANES 1999-2004 and 2011-2012.

Authors: Lopez DS1,2,, Advani S1,3, Qiu X1, Tsilidis KK4,5, Khera M6, Kim J7, Canfield S2.

Author Affiliations:1Deparment of Epidemiology, Human Genetics and Environmental Sciences, The University of Texas- School of Public Health, Houston, TX, USA2Division of Urology, UTHealth McGovern Medical School, Houston, TX, USA3Division of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington DC, USA4Department of Hygiene and Epidemiology, University of Ioannina School of Medicine, Ioannina, Greece5Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK6Scott Department of Urology, Baylor College of Medicine, Houston, TX, USA7Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Abstract (200 words)

Objective: The association of caffeine intake with testosterone remains unclear. We evaluated the association of caffeine intake with serum testosterone among American men and determined whether this association varied by race/ethnicity and measurements of adiposity.

Methods: Data were analyzed for 2,581 men (≥ 20 years old) who participated in the cycles of the NHANES 1999-2004 and 2011-2012, a cross-sectional study. Testosterone (ng/mL) was measured by immunoassay among men who participated in the morning examination session. We analyzed 24-h dietary recall data to estimate caffeine intake (mg/day). Multivariable weighted linear regression models were conducted.

Results: We identified no linear relationship between caffeine intake and testosterone levels in the total population, but there was a non-linear association (Pnonlinearity < 0.01). Similarly, stratified analysis showed nonlinear associations among Mexican-American and Non-Hispanic White men (Pnonlinearity ≤ 0.03 both) and only among men with waist circumference < 102 cm and body mass index < 25 kg/m2

(Pnonlinearity < 0.01, both).

Conclusion: No linear association was identified between levels of caffeine intake and testosterone in US men, but we observed a non-linear association, including among racial/ethnic groups and measurements of adiposity in this cross-sectional study. These associations are warranted to be investigated in larger prospective studies.

Keywords: caffeine intake, testosterone, race/ethnicity, obesity

Declaration of interest: the authors declare no conflict of interest.

Correspondence: David S. Lopez, Division of Epidemiology, Human Genetics and Environmental Sciences, University of Texas- Houston School of Public Health, 1200 Herman Pressler, Suite E-629, Houston, TX 77030, Phone: 713.500.6348, Fax: 713.500.9264 (fax), Email: David.S.Lopez@uth.tmc.edu

Introduction

Coffee has been implicated with a potential beneficial role against chronic diseases due to it

being a rich source of caffeine, antioxidants and anti-inflammatory compounds [1-3]. Recently, a large

prospective study reported that men who consumed six or more cups of coffee per day had a lower

adjusted relative risk for overall and lethal prostate cancer (PCa) compared with nondrinkers [4]. One

biological mechanism that has been hypothesized underlying this association is through the sex

steroid hormone-axis [4-6]. Although animal studies have suggested an inverse association between

caffeine and testosterone [7], yet the interplay between caffeine and testosterone levels in the

National Health and Nutrition and Examination Survey (NHANES) remains unclear.

Furthermore, a body of literature has identified that race and ethnicity and body fatness (body

mass index [BMI] and waist circumference [WC]) could influence the rates of PCa, with higher rates

being reported among African-Americans and obese men [8-11]. Interestingly, a line of research has

reported that testosterone levels are associated with race/ethnicity and body fatness [12-15]. These

studies have shown that Mexican American men had the highest levels of testosterone, whereas men

with large BMI and WC had lowest levels of testosterone. Surprisingly, there is a research gap in our

understanding on whether race/ethnicity and body fatness influence the association of caffeine intake

with testosterone levels. Therefore, the objectives of this study are to investigate the association of

caffeine intake with testosterone levels and determine whether this association varies by

race/ethnicity and body fatness.

Materials and Methods

Study Population

The National Health and Nutrition Examination Survey (NHANES) is a program of studies

undertaken by the National Center for Health Statistics (NCHS) of the US Centers for Disease Control

and Prevention (CDC) to assess the health and nutritional status of adults and children in the US.

NHANES uses a multistage, stratified and clustered probability sampling strategy in which Mexican-

Americans, non-Hispanic Blacks, and the elderly are oversampled to ensure adequate sample size

and to represent the total US civilian, non-institutionalized population [16]. The NHANES is a cross-

sectional study that includes an interview, and an examination component that includes blood

collection. Investigators are allowed to access surplus sera for approved studies. The present study

included data from male participants in the 1999–2000, 2001–2002, 2003–2004, and 2011-2012

NHANES cycles. Details of the survey design, methods and data collections are available on the

NHANES website (https://www.cdc.gov/nchs/nhanes/index.htm. Accessed 30 Jan. 2017).

Assessment of total testosterone

Total testosterone was measured from stored surplus serum samples by the study

investigators in 4,927 males, who were a stratified random sample of participants in the morning

examination sessions of each cycle. Morning sample participants were chosen to reduce extraneous

variation due to diurnal production of hormones. Male participants younger than 20 years were not

included in this study (n = 10,267). We excluded participants with a self-reported history of prostate

cancer because certain treatments may affect hormone levels leaving a final sample of 2,581 men.

The NHANES program is approved by the Institutional Review Board of the NCHS at CDC. Informed

consent was obtained from all participants. Addressing questions about hormones and men’s health

in NHANES was approved by the National Institutes of Health Office of Human Subjects Research

and the NCHS Ethics Review Board at CDC.

Details on the blood draw, process, storage and shipping methods are provided elsewhere [17,

18]. In NHANES 1999-2004, testosterone was measured using the Elecsys 2010 system (Roche

Diagnostics, Laval, QC, Canada). The laboratory technicians were blinded to the participant

characteristics of the samples. The lower limits of detection of the assays were 2 ng/dL for

testosterone. One sample had a concentration below the limit of detection for testosterone, which was

assigned to half the limit of detection (i.e. 1 ng/dL). Twenty-one samples were assayed in duplicate

for quality control purposes, and the coefficients of variation were 4.8% for testosterone. In NHANES

2011-2012, testosterone was measured with LC-MS/MS and was isolated from 100 μL serum by 2

serial liquid–liquid extraction steps and quantified with [13C] stable isotope–labeled testosterone as

the internal standard. The lower limit of detection was 0.3 ng/dL. Values of total testosterone below or

equal to 3.0 ng/mL are considered testosterone deficiency (TD) [19, 20]. In this study, the term TD

doesn’t imply that a deficit needs to be replaced, therefore, its use is analogous to low testosterone.

Assessment of caffeine and dietary data

The U.S. Department of Agriculture developed and validated a multiple-pass dietary recall

method for NHANES to collect dietary data [21]. Participants reported all food and beverages

consumed in two, 24-h dietary recall periods (midnight to midnight). The first one was conducted by

dietary research interviewers face-to-face, and the second one was done 3 to 10 days later by

telephone. Because NHANES 2001-2002 only included one recall and in order to maintain

consistency with the other cycles, our analysis was limited to the first-day dietary recall for NHANES

waves 2001-2002, 2003-2004 and 2011-2012. After the dietary interviews, USDA’s Food and Nutrient

Database for Dietary Studies 5.0 (2012) was used to code dietary intake data and calculate nutrient

intakes [21]. Based on the quantity of food and beverages reported and the corresponding nutrient

contents by the National Center for Health Statistics (NCHS), the caloric content and other nutrients

derived from each consumed food and beverage item were calculated [21, 22]. Data on caffeine

intake (mg/day), plain and tap water (gm), and alcohol (gm) was obtained from the Total Nutrient File,

which contains summed nutrients for an individual from all food and beverages provided on the

dietary recall [23]. We examined caffeine intake in six categories (mg/day), lowest category (C1; 0

mg/d), 2nd category (C2; 0.1-60 mg/d), 3rd category (C3; 61-200 mg/d), 4th category (C4; 201-325

mg/d), 5th category (C5; 326-450 mg/d), and 6th category (C6; ≥ 451 mg/d) using the International

Coffee Organization’s (http://www.ico.org/) information related to amount of caffeine per cup and

previous studies [24, 25]. Total water intake was categorized and defined by combining plain and tap

water, and it was included in multivariable models, while alcohol consumption was kept as continuous

variable [25, 26].

Assessment of covariates

Age, race/ethnicity, smoking status, education, diabetes and physical activity during the past

30 days (moderate and vigorous) were self-reported during the NHANES interview. NHANES

categorizes race/ethnicity as non-Hispanic white (NHW), non-Hispanic black (NHB) and Mexican

American (MA). Participants were classified as never, former and current smokers from self-reported

information; participants were asked if they had smoked more than 100 cigarettes in their lifetime and

if they were current smokers. Current smokers consisted of those who self-reported smoking habits

and smoked more than 100 cigarettes in their lifetime. Vigorous physical activity was obtained from

the questions on whether participants did any activity that caused heavy sweating or large increases

in breathing or heart rate (e.g., swimming, aerobics, or fast cycling), while moderate physical activity

was determined from the questions on whether they did any activities that caused light sweating or a

moderate increase in the heart rate, such as playing golf, dancing, bicycling for pleasure, or walking.

Body mass index (BMI) was calculated from measured weight and height (weight in kilograms divided

by height in meters squared). Overweight/obesity was defined by BMI ≥ 25 kg/m2. Waist

circumference (WC) was measured at the iliac crest. Abdominal obesity was defined as WC ≥ 102

cm. Type 2 diabetes status was defined as having fasting plasma glucose of ≥126 mg/dl, medication

treatment or being “told by a doctor you have diabetes or sugar diabetes.” Fasting plasma glucose

concentration was measured in the morning session after an overnight fast of at least 8 h [27], details

related to the laboratory procedures of that measurement is found elsewhere [16].

Statistical analyses

Sampling weights were applied to account for selection probabilities, over-sampling, non-

response, and differences between the sample and the total US population. Geometric means and

95% confidence intervals (CIs) for total testosterone concentrations were estimated by caffeine intake

using weighted linear regression models. Similarly, adjusted odds ratios (OR) and 95% CIs for TD

(≤ 3 ng/mL) using weighted logistic regression models were estimated in relation to six categorical

groups of caffeine intake. Total testosterone concentrations were transformed using natural logarithm

because they were right-skewed. Caffeine intake was categorized in six groups based on previous

studies [24, 25] including cut-off points of caffeine intake per cup provided by the International Coffee

Organization (http://www.ico.org/). To test for a linear trend across categories of caffeine intake, we

modeled caffeine intake as a continuous variable using the median for each category. To assess the

deviation from linear trend in a separate model, we included quadratic term for caffeine intake

(caffeine intake-squared) in addition to the continuous variable for caffeine intake and the p-value

associated with this quadratic term to report curvilinear associations [28]. Furthermore, a prior study

using NHANES caffeine data showed that caffeine intake did not follow a simple linear dose response

curve, but rather an exponential shape; therefore, we considered using the quadratic term will be

more appropriate in this association [25, 28, 29]. In the multivariable linear regression models, we

adjusted for age, race/ethnicity, education, BMI, smoking status, vigorous and moderate physical

activity, total water intake, total energy intake and total alcohol intake. Similarly, these confounders

were adjusted for in the multivariable logistic regression models to determine association between TD

and caffeine intake.

Stratified and multivariable analyses were conducted by race and ethnicity (NHW, MA, NHB)

and body fatness variables such as overweight/obesity (BMI ≥ 25kg/m) and abdominal obesity (WC ≥

102 cm) because these variables are known to modify testosterone levels. All p-values were two-

sided; alpha = 0.05 was considered the cut-off for statistical significance. All statistical analyses were

performed using STATA version 12.0 (College Station, TX).

Results

The distribution of baseline characteristics in the study population after applying sampling

weights is shown in Table 1. Fourteen percent of men reported 0 mg/day of caffeine intake, 28%

reported 61 to 200 mg/day of caffeine intake (equivalent to at least one cup of coffee per day), and

15% of men reported 201 to 325 mg/day of caffeine intake (equivalent to 2-3 cups of coffee). Eight

percent and 14% of men reported 326-450 mg/day and ≥451 mg/day, respectively, of caffeine intake.

As expected, in the last three categories of caffeine intake (> 201 mg/day) men tended to be older

(mean age >49 years old). Similar observations of high caffeine intake among older men were

detected when age was categorized as 20-30, 40-39, 40-49, 50-59 and ≥ 60 years old. Men with a

higher caffeine intake (> 201 mg/day) were more likely to be NHW, current smokers and non-diabetic.

In Table 2, mean value for caffeine intake was 226.0 mg/day, 2,010.0 mg/day for total water intake,

2,630.0 kcal/day for energy and 18.0 mg/day for alcohol.

In Figure 1, after adjusting for age, race/ethnicity, education, BMI, smoking status, vigorous

and moderate physical activity, total water intake, total energy intake and total alcohol intake, we did

not find a significant linear association between serum testosterone levels and categories of caffeine

intake (low to high) in the total population (Plinear trend = 0.21). However, we can’t exclude a possible

non-linear association (Pnonlinear < 0.01). It seemed that testosterone levels started decreasing as men

consumed 0.1-60 and 61-200 mg/day of caffeine intake. However, levels of testosterone increased

again among men with 201-325 mg/day of caffeine intake (equivalent of 2-3 cups of coffee), but there

was a subsequent decrease of testosterone in the two highest categories of caffeine intake, 326-450

and ≥ 451 mg/day.

Similarly, in total population, we investigated the association of TD (3.0 ng/mL of total

testosterone) with categories of caffeine intake (Figure 2). The category of 0 mg/day of caffeine intake

was selected as the reference group. In this analysis, there was no significant trend (Plinear trend = 0.78)

and also the independent comparisons between categories of caffeine intake with the reference

group (0 mg/day) were not significantly different: 0.1-60 mg/day (OR = 0.79, 95% CI: 0.49-1.30, P =

0.36), 61-200 mg/day (OR = 0.86, 95% CI: 0.54-1.39, P = 0.54), 201-325 mg/day (OR = 0.91, 95%

CI: 0.62-1.33, P = 0.62); 326-450 mg/day (OR = 0.89, 95% CI: 0.43-1.81, P = 0.73) and ≥ 451 mg/day

(OR = 0.78, 95% CI: 0.42-1.45, P = 0.43).

The association of serum testosterone levels with caffeine intake was further investigated by

stratification with race and ethnicity (Figure 3) and body fatness (Figures 4) in multivariable analysis.

Among MA men, we identified a significant non-linear U-shape association between caffeine intake

(low to high) and testosterone levels (Pnonlinear = 0.03). Mean levels of testosterone from categories of

caffeine intake, 0.1-60 mg/day and 61-200 mg/day, were significantly different from mean level of

testosterone (6.61 ng/mL) in men with 0 mg/day of caffeine intake, 5.91 ng/mL (95% CI: 5.52-6.15, P

= 0.03) and 5.92 ng/mL (95% CI: 5.60-6.25, P = 0.002), respectively. Among NHW men, we did not

find a linear association between caffeine intake and testosterone (Plinear trend = 0.55), but there was a

significant non-linear association (Pnonlinear < 0.01). No significant associations of any shape were

found among NHB men.

In Figure 4, we stratified the association between caffeine intake and testosterone levels by

overweight/obesity (BMI ≥ 25 kg/m2). No linear relationship between caffeine intake and testosterone

was identified in any of the two groups, non-overweight/obesity and overweight/obesity, Plinear trend =

0.47 and 0.34, respectively. However, we identified a nonlinear association between caffeine intake

and testosterone levels only among men in the non-overweight/obesity group (Pnonlinear < 0.01).

Stratification by abdominal obesity (WC ≥ 102 cm) showed similar nonlinear association in the non-

abdominal obesity group (Pnonlinear < 0.01) (Supplemental Data Figure 1). Finally, it was observed that

men who were in the overweight/obesity and abdominal obesity groups had lower levels of

testosterone than those men who were in the non-overweight/obesity and non-abdominal obesity

groups.

We also analyzed the association of caffeine intake (continuous variable) with serum

testosterone, including stratification by race/ethnicity, and testosterone deficiency. The

results are presented in an Online Supplement (Supplemental Data Tables 1-3). In general, we

did not find any statistical significant associations. Similarly, stratification of the association

between caffeine intake and testosterone by waist circumference was not significant and did

not convey any different message than stratification by BMI (Supplemental Data Figure 1.)

Discussion

In this cross-sectional study of the NHANES cycles 1999-2004 & 2011-2012, we examined

the association of caffeine intake with serum testosterone levels and determined whether this

association varied by race and ethnicity and measurements of adiposity. These results showed that

there was no linear relationship between caffeine intake and testosterone levels in the total

population, but we can’t exclude a potential non-linear association. When the outcome was defined

as TD (≤ 3.0 ng/mL of total testosterone), the association with caffeine intake was not significant as

well. In stratified analysis, only in MA men there was a significant association between caffeine intake

and testosterone levels and a significant non-linear J-shape association. In NHW men, we identified a

potential nonlinear association, and null results were reported in NHB men. When the association of

caffeine intake with testosterone levels were stratified by overweight/obesity (BMI ≥ 25 kg/m2), only in

those men who were not overweight/obese we found significant associations.

The relationship between caffeine intake and testosterone levels has been previously

investigated in women, but surprisingly very few studies have been conducted among men [6, 30-33].

However, with the exception of one study [33], most of these studies conducted among men have to

be interpreted with caution mainly due, in part, to their small sample size. McDonald et al. showed an

increased in testosterone concentration in healthy male amateur games players (n=11), who were

sleep-deprived, after caffeine supplementation (6 mg.kg-1, an equivalent of approximately 3 cups of

coffee) [30]. Wu et al. showed an increased in testosterone concentration after a high-caffeine dose

(6 mg.kg-1) in 12 university male athletes [31]. Wedick et al. in a parallel-arm randomized controlled

trial of 14 healthy men reported that consumption of caffeinated coffee (five 6-ounce cups of

caffeinated coffee) increased total testosterone at week 4, but not at week 8 [6]. Other randomized,

double-blind, placebo-controlled, balance trial of 24 men showed an increase of testosterone in a

dose-dependent manner by high concentration of caffeine (800 mg) [32]. A previous large European

cross-sectional study of 1,563 men showed a positive association between coffee (cups/day) and

total testosterone, however, statistical significance disappeared when low vs high number of cups of

coffee were compared (1-4 cups vs >4 cups of coffee) [33].

In a similar way, there is also a limited research on addressing the independent association of

caffeine intake with testosterone levels stratified by race/ethnicity and measurements of adiposity [5,

33]. The significance to conduct this type of investigation is that previous studies have reported

independent associations of testosterone with race/ethnicity and measurements of adiposity in

NHANES [12-15]. Therefore, the investigation of the association between caffeine intake and

testosterone levels stratified by race/ethnicity and measurements of adiposity is warranted.

Several aspects of our findings merit discussion. First, we did not find a significant linear

association between caffeine intake and testosterone levels in the total population, which is in

disagreement from previous studies conducted among men [6, 30-33]. One of the main differences

between previous studies and the current study is the sample size. Our study included 2,581 men

whose age was ≥ 20 years old. Some of the benefits of a large sample size are that they are more

representative of the population, limiting the influence of outliers and extreme observations, and an

increase in precision. In contrast, the two previous randomized trials [6, 32] can minimize the effects

of unknown confounders in the association between caffeine intake and testosterone levels, which is

difficult to address in a cross-sectional study irrespective of the large sample population. Although

another previous cross-sectional study reported a positive association between cups of coffee and

total testosterone, this significance was attenuated when they compared 1-4 cups vs >4 cups of

coffee thus concurring with our null findings in the total population [33]. Second, although we can’t

exclude a non-linear association between caffeine intake and testosterone, we will interpret this

finding with caution for the following reasons, a) it may be due to chance and b) there is still no well-

established biological plausibility about the differential effects of low-, medium- or high-caffeine intake

on the regulation of testosterone production. However, the mechanistic explanations for nonlinear

patterns in caffeine intake-testosterone associations are complex yet plausible, in part because

different processes likely explain different aspects of the findings. Future prospective and larger

studies could focus on whether different amounts of caffeine have differential effects on testosterone

levels or its production. Third, comparison of caffeine intake among studies is not standardized. Our

study included total caffeine intake obtained from all food and beverages reported in the 24-h dietary

recalls, and the categorical groups of caffeine intake per day presented in this study only provided

approximation of cups of coffee per day following cut-off points of caffeine intake provided by the

International Coffee Organization (http://www.ico.org/) and previous studies [24, 25]. Moreover, the

two randomized trials reported significant associations with high caffeine intake (800-mg dose)[32]

and a high intake of cups of coffee (five 6-ounce cups of caffeinated coffee)[6], but this high intake of

caffeine or cups of coffee is not typical in the general population. The other studies reported an intake

of 6 mg.kg-1 of caffeine supplementation (equivalent of approximately 3 cups of coffee) [30, 31]. Yet it

is worthwhile to mention that these previous studies were conducted in more controlled settings for

caffeine intake. Therefore, it is possible that the effects of caffeine on testosterone in controlled

settings may be different than the ones obtained from 24-dietary recalls in cross-sectional studies.

Fourth, we couldn’t adjust for sex-hormone binding globulin and estradiol levels because they were

not included in the latest NHANES 2011-2012 wave, and we wanted to capitalize on the large sample

size by combining these two NHANES waves. Fifth, our multiple linear regression models included

confounders, such as total water intake and total energy, that have not been previously included in

other studies, but they have the potential to mask the association between caffeine intake and

testosterone levels. Finally, stratification analysis reduces the sample size in both the exposure

and the outcome, and to a greater extent in categorical variables, therefore, the significant

associations identified in this stratified analyses may be due to chance and they should be

interpreted with caution.”

Similar unexpected results were identified after stratifying by measurements of adiposity

showing no significant linear associations of caffeine intake with testosterone, but only possible

nonlinear associations among men who were in the non-overweight/obesity and non-abdominal

obesity groups. Furthermore, irrespective of caffeine intake, men who were included in the

overweight/obesity and abdominal obesity groups had lower levels of testosterone than those men

who were in the non-overweight/obesity and non-abdominal obesity groups. It remains unclear what

biological mechanism (s) may be influenced in this latter observation. Thus, future large prospective

studies may address this observation.

Our study has many strengths. NHANES is a program of studies that is representative of the

civilian noninstitutionalized US population, which aids in the generalizability of these results. In

addition, NHANES follows a rigorous protocol with extensive quality control procedures for the

collection of the exposures (i.e. validated dietary recall methodology [21, 22]), outcome of interest and

potential confounding factors analyzed and adjusted in this study. Despite these strengths, the

current study has several limitations, which may influence the interpretation of these results. First, we

relied on a single measurement of total testosterone. Second, although we adjusted for several

potential confounders, there is still the possibility of unmeasured confounding from additional factors.

However, due to our detailed adjustment for confounders, it is unlikely these would fully account for

the observed findings. Finally, NHANES is a cross-sectional study, and there is an inherent bias in

the use of surveys for data collection. Thus, these findings should be confirmed in large prospective

studies.

Conclusions

In general, we did not find significant linear association between caffeine intake and total

testosterone in the total population, but we can’t exclude potential nonlinear associations.

Stratification analyses by race/ethnicity and measurements of adiposity seemed to influence the

association of caffeine intake with total testosterone. Yet, the role of race/ethnicity and measurements

of adiposity in these associations should be investigated in larger prospective studies.

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33. Svartberg J, Midtby M, Bonaa KH, et al. The associations of age, lifestyle factors and chronic disease with testosterone in men: the Tromso Study. Eur J Endocrinol 2003;149:145-152. 

Table 1. Selected characteristics of the U.S. Population of adult men aged 20 years and older- NHANES 1999-2004 and 2011-2012

Caffeine intake, mg/daya

Characteristicsa 1st Category (0 mg/d caffeine, n = 362)

2nd Category (0.1 – 60 mg/d caffeine, n = 540)

3rd Category (61 – 200 mg/d caffeine, n = 735)

4th Category (201 – 325 mg/d caffeine, n= 386)

5th Category (326 – 450 mg/d caffeine, n = 198)

6th Category (≥ 451 mg/day caffeine, n = 360)

P-Value

Age (y), mean (range) 40.81(21-85)

45.88(21-85)

46.17 (21-85)

49.71(21-85)

52.77(22-85)

49.66(21-85)

<0.001

Age categories, (%) <0.001 20-39 y

40-49 y

50-59 y

≥60 y

177(55.08%)

57(15.75%)

45(16.83%)

83(12.34%)

195(42.95%)

78(19.03%)

64(13.53%)

203(24.49%)

257(40.55%)

115(19.08%)

104(16.27%)

259(24.1%)

100(25.9%)

78(22.02%)

82(27.44%)

126(24.64%)

49(27.16%)

31(19.07%)

45(25.15%)

73(28.62%)

95(27.11%)

73(25.73%)

68(22.91%)

124(24.25%)

Race/ethnicity, (%) <0.001 Mexican Americans 69

(14.93%)94

(10.46%)165

(12.32%)67

(7.65%)23

(3.74%)44

(5.3%) Non-Hispanic white 109

(56.39%)242

(73.79%)374

(76.80%)252

(86.56%)158

(93.84%)243

(88.59%) Non-Hispanic black 184

(28.68%)204

(15.74%)196

(10.88%)67

(5.79%)17

(2.42%)73

(6.11%)Education, (%) 0.07 Less than 9th grade 55

(8.25%)71

(6.28%)89

(5.47%)42

(5.26%)16

(4.91%)38

(6.55%) 9-11th grade 71

(13.07%)79

(10.92%)103

(10.20%)65

(11.7%)23

(7.65%)64

(15.72%) High school graduate/GED or equivalent

74(20.51%)

127(19.93%)

162(19.88%)

94(23.2%)

52(29.5%)

89(26.11%)

Some college or AA degree

105(32.64%)

146(27.25%)

205(30.0%)

96(28.89%)

63(31.02%)

98(27.0%)

College graduate or above

57(25.53%)

117(36.16%)

175(34.45%)

89(30.95%)

44(26.92%)

71(24.62%)

Body Mass Index (kg/m2), (%)

0.46

Normal (≤ 24.9)

Overweight (≥ 25 - 29.0)

Obese (≥ 30)

110(29.2%)

117(32.77%)

132(38.03%)

142(27.46%)

200(39.82%)

185(32.73%)

177(24.5%)

286(40.71%)

262(34.79%)

100(26.24%)

151(39.28%)

130(34.48%)

52(25.72%)

80(43.68%)

62(30.60%)

94(23.69%)

127(37.65%)

135(38.66%)

Smoking Status, (%) <0.001 Never Smoker

Former Smoker

Current Smoker

176(53.09%)

54(12.98%)

131(33.93%)

262(57.07%)

143(20.52%)

134(22.41%)

313(47.34%)

207(25.59%)

214(27.08%)

131(36.62%)

117(31.13%)

138(32.25%)

53(30.52%)

59(29.79%)

86(39.69%)

97(30.08%)

92(23.38%)

171(46.54%)

Vigorous Physical Activity, (%)

0.65

Yes

No

184(43.72%)

175(56.28%)

271(48.82%)

262(51.18%)

363(48.99%)

367(51.01%)

176(44.02%)

206(55.98%)

101(51.04%)

94(48.96%)

172(46.78%)

185(53.22%)

Moderate Physical Activity, (%)

0.79

Yes

No

150(38.15%)

209(61.85%)

205(37.06%)

329(62.94%)

291(40.57%)

443(59.43%)

146(32.37%)

239(67.63%)

80(42.11%)

117(57.89%)

150(39.02%)

207(60.98%)

Type 2 Diabetes, (%) 0.06 Yes

No

49(11.58%)

208(88.42%)

93(13.08%)

305(86.92%)

113(16.16%)

402(83.84%)

55(12.5%)

229(87.5%)

41(22.20%)

91(77.80%)

65(22.22%)

189(77.78%)

a Sampling weights were applied

Table 2. Summary of dietary characteristics for NHANES cycles 1999-2004 and 2011-2012

Dietary characteristics N Meana 95% CIa

Total caffeine, mg/day 2,455 226.00 (204.20 - 247.50) Total water, gm/day 2,455 2,010.00 (1,837.79 - 2,181.76) Total energy, kcal/day 2,455 2,630.00 (2,575.72 - 2,683.36) Alcohol, gm/day 2,455 18.00 (15.25 - 20.49)

a Sampling weights were applied

Figure 1. Geometric means and 95% confidence intervals of total testosterone (ng/mL) concentrations by categories of caffeine intake in 2,581 participants (≥ 20 years old) in the NHANES cycles 1999-2004 and 2011-2012

5.7

6.2

6.7

7.2

7.7

8.2

8.7

9.2

9.7

10.2

10.7

Categories of caffeine intake (mg/day)

Geo

met

ric M

ean

and

95%

CI

Pnonlinearity < 0.01Plinear trend = 0.14

Multivariable model was adjusted for age, race/ethnicity, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake.*Significantly different from 0 mg/day, P < 0.05.

Figure 2. Association† of caffeine intake categories with testosterone deficiency (≤ 3 ng/mL) in the NHANES cycles 1999-2004 and 2011-2012

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Categories of caffeine intake (mg/day)

OD

DS

RA

TIO

Pnonlinearity = 0.48Plinear trend = 0.78

†Model adjusted for age, race/ethnicity, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake.*Significantly different from 0 mg/day, P < 0.05.

Figure 3. Geometric means and 95% confidence intervals of total testosterone (ng/mL) concentrations by categories of caffeine intake stratify by race and ethnicity: NHANES cycles 1999-2004 and 2011-2012

3.5

4.5

5.5

6.5

7.5

8.5

9.5

Categories of caffeine intake (mg/day)

Geo

met

ric M

ean

and

95%

CI

MA

NHW

NHB

Pnonlinearity = 0.03Pnonlinearity < 0.01

Pnonlinearity = 0.99Plinear trend = 0.009

Plinear trend = 0.55Plinear trend = 0.64

* *

*

Multivariable model was adjusted for age, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake. MA = Mexican-Americans; NHW = non-Hispanic white; NHB = non-Hispanic black. C1= 0 mg/day; C2 = 0.1-60 mg/day; C3 = 61-200 mg/day; C4 = 201-325 mg/day; C5 = 326-450 mg/day; C6 = ≥451 mg/day.*Significantly different from 0 mg/day, P < 0.05.

Figure 4. Stratified by body mass index (≥ 25 kg/m2) the association of caffeine intake categories with total testosterone (ng/mL) concentrations in 2,581 participants (≥ 20 years old) in the NHANES cycles 1999-2004 and 2011-2012

5

5.5

6

6.5

7

7.5

8

8.5

9

Categories of caffeine intake (mg/day)

Geom

etric

Mea

n an

d 95

% C

I

BMI < 25 kg/m2

BMI ≥ 25 kg/m2

Pnonlinearity < 0.01

Pnonlinearity = 0.52

Plinear trend = 0.47

Plinear trend = 0.34

Multivariable model was adjusted for age, education, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake. C1= 0 mg/day; C2 = 0.1-60 mg/day; C3 = 61-200 mg/day; C4 = 201-325 mg/day; C5 = 326-450 mg/day; C6 = ≥451 mg/day. Body mass index ≥ 25 kg/m2 is defined as overweigh/obesity.*Significantly different from 0 mg/day, P < 0.05.

Supplemental Data Table 1. Association of caffeine intake (continuous, mg/day) with total testosterone (ng/mL) in 2,581 participants (≥ 20 years old) in the NHANES cycles 1999-2004 and 2012

Model 1 Model 2ᵝ Pvalue ᵝ Pvalue

Caffeine intake (mg/day)

0.0031 0.644 0.0030 0.600

Model 1: age-adjustedModel 2: Multivariable model was adjusted for age, race/ethnicity, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake.

Supplemental Data Table 2. Association of coffee intake (continuous, mg/day) with testosterone deficiency (≤ 3 ng/mL) in the NHANES cycles 1999-2004 and 2011-2012

Model 1 Model 2OR 95% CI OR 95% CI

Caffeine intake (mg/day)

1.006 0.901, 1.122 0.996 0.897, 1.105

Model 1: age-adjustedModel 2: Multivariable model was adjusted for age, race/ethnicity, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake.

Supplemental Data Table 3. Association of caffeine intake (continuous, mg/day) with total testosterone (ng/mL) stratified by race and ethnicity.

Full-adjusted Model Race and Ethnicity

ᵝ Pvalue

Mexican-Americans Caffeine intake (mg/day)

-0.002 0.983

Non-Hispanic Whites Caffeine intake 0.0067 0.393

(mg/day)Non-Hispanic Blacks Caffeine intake (mg/day)

-0.0015 0.923

Full-adjusted model: model was adjusted for age, education, BMI, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake.

Supplemental Figure 1. Stratified by waist circumference (≥102 cm) the association of caffeine intake categories with total testosterone (ng/mL) concentrations in 2,581 participants (≥ 20 years old) in the NHANES cycles 1999-2004 and 2011-2012

4.5

5

5.5

6

6.5

7

7.5

8

Categories of caffeine intake (mg/day)

Geom

etric

Mea

n an

d 95

% C

I

Waist circumference < 102 cm

Waist circumference ≥ 102 cm

Pnonlinearity < 0.01

Pnonlinearity = 0.60

Plinear trend = 0.03

Plinear trend = 0.95

Multivariable model was adjusted for age, education, smoking status, vigorous and moderate physical activity, total water intake, total energy intake and total alcohol intake. C1= 0 mg/day; C2 = 0.1-60 mg/day; C3 = 61-200 mg/day; C4 = 201-325 mg/day; C5 = 326-450 mg/day; C6 = ≥451 mg/day. Waist circumference ≥ 102 cm is defined as abdominal obesity.

*Significantly different from 0 mg/day, P < 0.05.