Abstract
Background:
Testosterone (T) plays a crucial role in various physiological functions in men, and understanding the variations in T levels during the day is essential for diagnosing and treating testosterone deficiency (TD).
Aim:
We sought to evaluate the reduction in serum total T (TT) levels throughout the day in men with symptoms of testosterone deficiency and to determine the variables having an impact on the extent of this decline.
Methods:
The study population consisted of a group of men who within 3 months of each other had all undergone both early morning and afternoon TT level measurements. We did not include patients with a history of a prior orchiectomy, testosterone levels below 100 ng/dL or above 1000 ng/dL, a history of androgen deprivation therapy, or patients on T therapy. Statistical analyses were conducted using descriptive statistics, t-tests, chi-square tests, and correlation calculations. Liquid chromatography–tandem mass spectrometry was used to measure TT, and a change in TT levels greater than 100 ng/dL was considered significant. Using multivariable and univariable analysis, we attempted to define predictors of a decrease in afternoon TT levels.
Outcomes:
The majority of men showed no significant difference in T levels between morning and afternoon.
Results:
In total, 506 men with a median age of 65 years were analyzed. The most common comorbidities were hypertension and hyperlipidemia. Levels of TT were measured in the morning and afternoon, and no significant differences in mean T levels based on the time of the test were found. Age was not significantly associated with T levels.
Clinical Implications:
There was a weak negative correlation between age and the difference between morning and afternoon T levels, with younger men showing more significant variations in T levels. The most considerable differences in T levels were observed in men younger than 30 years. There were no predictors of the magnitude of the T decrease in the afternoon.
Strengths and Limitations:
Strengths of the study include the number of subjects and the use of liquid chromatography–tandem mass spectrometry for T measurement. Limitations include failure to measure morning and afternoon T levels on the same day, the retrospective nature of the study, and a smaller sample size of patients younger than 30 years.
Conclusion:
In this study we found no strong link between age and daily T fluctuation, but we observed a decrease in the magnitude of variation with aging. The group experiencing the most significant decline in daily T had higher morning and consistently normal afternoon T levels.
Keywords: testosterone, diurnal variation, circadian rhythm, testosterone deficiency
INTRODUCTION
Testosterone (T) is involved in multiple physiological functions in the body and is the most common androgen in males. T production is regulated by the hypothalamic-pituitary-gonadal axis Gonadotropin-releasing hormone (GnRH) produced by the hypothalamus stimulates the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the anterior pituitary gland. LH stimulates the Leydig cells in the testes to produce T. (1–4) T is secreted into the blood, which can vary with the circadian rhythm. The predominantly circulating total T is bound to albumin and sex hormone-binding globulin (SHBG), and a small percentage is circulating freely, only 1–2%. It is free T that has endocrine effects on the tissues and cells or undergoes conversion to downstream hormones estradiol or dihydrotestosterone. T exerts its physiological functions through androgen receptors (AR). After binding with AR, this complex is transferred to the cell nucleus, where it will respond to androgenic response elements in DNA, where androgen-dependent genes will be expressed or repressed that will eventually act on target tissues. (5–7) Both have essential and differential roles in various tissues, which include the penis, testicles, and prostate. (8–15)
Low serum T combined with specific symptoms and signs is a clinical syndrome called testosterone deficiency (TD). The most common symptoms and signs are low sex drive, fatigue, depressed mood, erectile dysfunction, reduced muscle mass, increased fat mass, and reduced bone density. (16, 17) The prevalence of TD in a population of men can vary depending upon the criterion used to define low T, with estimates in excess of 70% in some populations. (18) TD diagnosis and treatment have increased significantly over the past decade, with an important rise in T prescription rates. (19–21) Given the increasing number of diagnoses, specialists managing TD must understand the methodology and limitations of the available tools for the diagnosis.
T is secreted in pulses 8 – 14 times a day, and the understanding of testosterone variation during the day is crucial for the recognition of TD and its treatment. (2) Serum testosterone concentrations vary throughout the day, influenced by the circadian rhythm, with peak levels occurring in the morning and lower in the afternoon. (2, 22) The highest levels are between 5–8 am, and the lowest levels are between 6–11 pm, with a reported 10–25% decrease over the day.(23–27) Based on this variation, the AUA guidelines do not recommend measuring serum testosterone after 10 am.(28) This diurnal variation in testosterone secretion is higher in young men and seems to be reduced in older men.(23, 24, 26, 29, 30) There is little data on the magnitude of the afternoon drop in T levels, and in clinical practice, we see many older men with a significant diurnal variation. Therefore, our study aimed to define the magnitude of the decrease in serum total testosterone levels over the day and whether any factors predicted the magnitude of such a drop.
METHODS
Study Population:
After receiving approval from the institutional review board (IRB 16–1526), we examined our institution’s database for male individuals who presented with low T symptoms and had undergone blood collection for total testosterone measurement both in the early morning (before 10 am) and in the afternoon (after 2 pm) within a maximum of three-month interval. We then paired these individuals and collected data on their demographics and medical conditions, including hyperlipidemia, hypertension, diabetes mellitus, obstructive sleep apnea, coronary artery disease, peripheral vascular disease, and smoking status. To ensure the validity and avoid any contamination from patients, we excluded patients who had undergone a prior orchiectomy, those with testosterone levels below 100 ng/dL or above 1000 ng/dL, individuals with a history of androgen deprivation therapy, and patients currently undergoing testosterone replacement therapy.
Testosterone Measurement:
All total testosterone tests were performed using liquid chromatography-tandem mass spectrometry (LCMS). LCMS has excellent sensitivity and specificity at low testosterone concentrations and has the lowest coefficient of variation compared to the other assays, estimated to be around ±6.4%, in contrast to ±15% for immunoassays and 13% for the analog methods. For this reason, the AUA guidelines panel recommends that LCMS be the gold standard for measuring total testosterone levels.(28, 31)
Statistical Analyses:
Descriptive statistics are reported as median (IQR) for continuous variables and percentages for categorical variables. To statistically compare the groups, continuous variables were assessed with a 2-tailed Student’s T-test and categorical variables by chi-square test. We report the directionality of the changes based on increases or decreases in T levels between morning and afternoon. No significant difference was defined as a delta in total testosterone levels from −100 to + 100 ng/dL, with significant changes >100 ng/dL in either direction. ANOVA analysis was used to seek differences in these three groups. The correlation between total testosterone level decrease over the day and age was calculated. Finally, we attempted using multivariable analysis to define predictors of a decrease in afternoon TT level of >100 ng/dl. Univariable analysis was performed to define predictors of such a decrease. The factors explored included: patient age, presence of diabetes, number of vascular comorbidities (1–5, including hypertension, hyperlipidemia, cigarette smoking, obstructive sleep apnea and coronary artery disease), baseline TT level. All factors significant at <0.20 level moved forward into the multivariable model. A p value of < 0.05 was considered statistically significant.
RESULTS
Patient Population:
The study population included 570 men. After exclusions, a total of 506 men were available for analysis. The median age was 65 (57, 70), with 6% younger than 40, 30% between 40–60, and 64% older than 60. The most common comorbidities were hypertension 43%, hyperlipidemia 42%, obstructive sleep apnea 14% and 5% were current smokers (Table 1).
Table 1 –
Patient Demographics
| Variable | Results | |
|---|---|---|
| Age, median (IQR; years) | 64 (57, 70) | 354 |
| Hypertension, n (%) | 217 (43) | |
| Hyperlipidemia, n (%) | 210 (42) | 355 |
| Obstructive Sleep Apnea, n (%) | 72 (14) | |
| Diabetes Melitus, n (%) | 41 (8) | --- |
| Coronary Arterial Disease, n (%) | 25 (5) | |
| Peripheral Vascular Disease, n (%) | 1 (0) | 358 |
| ≥ 2 comorbidities, n (%) | 169 (33) | |
| Current smoker, n (%) | 24 (5) | 359 |
IQR = interquartile range
Testosterone Levels:
Morning total T levels were measured before 10 am, and afternoon tests were collected after 2 pm. The median of the morning total T was 387 (306, 505) ng/dL, and the afternoon was 373 (281, 486) ng/dL (no statistically significant difference). 63% of men had no significant difference between morning and afternoon levels (−100 ng/dL to +100 ng/dL), but 24% had a decrease of over 100 ng/dl, and 13% had an increase of more than 100 ng/dl. We did not find significant differences when comparing afternoon T levels obtained after 2 pm, 4 pm, or 6 pm. Neither morning nor afternoon total T levels were significantly associated with age. However, age was weakly negatively correlated with the magnitude of the difference between morning and afternoon levels (r = 0.10, p = 0.03), with more significant variation seen in younger men (Figure 1b).
Figure 1:
(A) Graph of median total testosterone variation between morning vs. afternoon distributed by the decade; B) Scatter plot graph of total testosterone variation between morning vs. afternoon by age (years).
Upon comparing total T levels between morning and afternoon by age decade, it is noteworthy that the most disparity was observed in individuals under 30, with a negative variation of 79ng/dL and no significant changes in the older population, as illustrated in Figure 1a. A comprehensive overview of the testosterone levels by decade of age is presented in Table 2.
Table 2 –
Total Testosterone Levels Distributed by Age
| Age (years) | No. (%) | Morning Median TT (IQR) | Morning Median TT (IQR) |
|---|---|---|---|
| ≤ 30 | 12 (2) | 462 (316–559) | 383 (292–446) |
| 31–40 | 21 (4) | 403 (329–489) | 400 (323–489) |
| 41–50 | 31 (6) | 370 (331–570) | 370 (258–511) |
| 51–60 | 117 (23) | 356 (292–499) | 350 (265–451) |
| 61–70 | 208 (41) | 388 (315–502) | 374 (291–464) |
| >70 | 117 (23) | 403 (298–497) | 394 (291–514) |
TT = Total Testosterone; IQR = interquartile range.
We observed that among men with low testosterone levels (<300 ng/dL) in the afternoon, 14% showed normal testosterone levels during the morning. Additionally, we observed that the group of men who experienced the most substantial decline in total testosterone levels in the afternoon (>100 ng/dL) had higher total testosterone levels in the morning, with a median of 548 (444, 713) ng/dL. On the other hand, the group of men with no significant variation in total testosterone levels (−100 ng/dL to +100 ng/dL) had lower morning levels, with a median of 349 (286, 445) ng/dL, as shown in Table 3.
Table 3 –
Groups Categorized by Magnitude of Total Testosterone Level Variation
| Group 1 | Group 2 | Group 3 | P Value | |
|---|---|---|---|---|
| Population, n (%) | 322 (63) | 120 (24) | 64 (13) | - |
| Age, median (IQR) | 65 (58, 70) | 62 (55,68) | 64 (58,69) | 0.23 |
| Morning TT, median (IQR) | 349 (286, 445) | 548 (444, 713) | 368 (294, 442) | 0.01 |
| Afternoon TT, median (IQR) | 347 (272, 439) | 374 (255, 449) | 566 (480, 648) | 0.01 |
Group 1: TT no significant difference between am and pm levels (−100 to + 100 ng/dL).
Group 2: decrease in TT level in pm (−100 ng/dL).
Group 3: increase in TT level in pm (+100 ng/dL).
TT = Total Testosterone; IQR = interquartile range.
The only predictors of a decrease of >100 ng/dl TT level in the afternoon were patient age (OR 1.5, 95% CI 1.2–3.4, p<0.01) and baseline TT >400 ng/dl (OR. 1.2, 95% CI 1.1–1.9).
DISCUSSION
In recent decades, numerous studies have demonstrated that older men have lower testosterone levels and in contemporary medicine this is appreciated to be the result of a diminution of the early morning T surge, which is confirmed by this current analysis. (23, 32–40) Some studies have observed in healthy men that the T levels do not decrease significantly with age.(41–43) Harman et al. support reducing T levels with aging at a constant rate, independent of smoking, alcohol intake, obesity, comorbidities, and medication. They measured the T levels using radioimmunoassay in 890 healthy American men in the Baltimore Longitudinal Study on Aging and found that the TD rate increased with age, observing about 20% of men over 60, 30% over 70, and 50% over 80 years old.(44) Boyce et al. studied 266 healthy men between 18 and 70 years old, with measures of total T in the morning (8–9 am) and evening (8–9 pm). They reported a significant decline between morning and evening serum testosterone levels, with a mean total T of 502 ng/dL in the morning and a mean of 404 ng/dL in the evening. However, no significant variation among the men over 60 was detected between morning and evening T levels. (45)
Our study found a slight difference between morning and afternoon T measurements, a median of 387 (306, 505) ng/dL in the morning and 373 (281, 486) ng/dL in the afternoon, with no significant difference. Age was not a predictive factor in changes in serum testosterone levels between morning and afternoon. Brambilla et al. found in 11 men between 30 and 40 years old that the total T was 30 – 35% higher in the morning than in the afternoon. Although this difference decreased among older men, dropping to nearly 10% at 70.(46) In contrast, our study with an older population, with a median age of 64 years, showed no significant difference in the measures in 63% of the men. Bremner et al. analyzed the testosterone levels within 24 hours in 17 young men, with a mean age of 25 years old, and twelve older men, with a mean age of 71 years old. They were of average weight, non-smokers, non-alcohol abusers, and without medication. It was observed that the morning rise of testosterone was presented in young men but not in the older, and in the young men the testosterone levels during the 24 hours were higher than in the older men.(23)
Similar to our finding, Crawford et al. analyzed the testosterone levels of 3006 men, mean age of 60, which were submitted to a prostate cancer screening. The study population was divided into groups based on the blood test time (6 am to 6 pm) and age (decades of life). The mean testosterone level was 415 ng/dL. All groups had a mean decline of 13% in testosterone levels after 2 pm. Another important finding is that men with low testosterone (≤300 ng/dL) did not significantly differ in the mean levels of total testosterone between morning and afternoon(40). Plymate et al. analyzed ten healthy young men (mean age of 27) and ten healthy older men (mean age 70), and all of them had normal testosterone levels (>280 ng/dl). They did the blood test between 8 – 10 am and 8 – 9 pm for 24 hours.(27) They observed a reduction of the amplitude in the older man group, consistent with previous studies, with lower variability when the age increases.(23–27, 47) The reduction of total T amplitude between morning and afternoon in older men was observed in our study. We also observed a rise of at least 100 ng/dL in testosterone level after 2 pm in 13% of the men. Similar to our research results, Brambilla et al. (46) observed that men exhibited low testosterone levels before noon, but their testosterone levels were within the normal range (>300ng/dL) during the afternoon. The authors postulated that this disparity could be attributed to measurement variability, which sets our study apart from theirs. Notably, they employed the Radioimmunoassay method, while we used LCMS, which demonstrated a reduced coefficient of variation.
When examining the testosterone levels in different age groups, we observed that individuals under 30 showed a higher degree of difference in testosterone levels between morning and afternoon. It is important to note that this specific subgroup accounted for only 2% of the total number of participants in our study. Our findings are consistent with the research conducted by Boyce et al., who also discovered a negative relationship between age and testosterone concentrations. They found that younger males under 40 had more significant variations in testosterone levels between morning and evening, whereas older groups exhibited less variability in this aspect.
The diagnosis of TD requires low T(<300ng/dL) and association with signs and symptoms. The blood test timing might be significant given the variation of testosterone according to circadian rhythm. However, we did not find a relevant variation in testosterone levels in 63% of the men, and when the testosterone levels decreased more than 100 ng/dL between am/pm, the patients had higher morning levels, a median of 548 (444, 713) ng/dL, with normal levels in the afternoon, a median of 374 (255, 449) ng/dL.
Therefore, considering this and the previous studies, it may not be highly relevant to collecting the morning measurement of testosterone for diagnoses of TD, and we could have a longer window time opportunity to collect the exam. Although T values in morning samples are more reliable for diagnosis of hypogonadism, normal T values from afternoon blood samples may be helpful in excluding hypogonadism.
Our strengths include the number of subjects and the use of LCMS for testosterone measurement with the LCMS method. Our limitations include failure to measure morning and afternoon T levels on the same day, the retrospective nature of the study, and a smaller sample size of patients below 30 years.
CONCLUSIONS
Our findings show no significant association between age and degree of testosterone variation during the day. However, the magnitude of variation decreases with aging, and the group with the most significant decrease during the day (>100ng/dL) had the highest morning testosterone levels and routinely normal T levels in the afternoon.
Source of funding:
Sidney Kimmel Center for Prostate and Urologic Cancers and the National Institutes of Health National Cancer Institute to Memorial Sloan Kettering Cancer Center through the Cancer Center Support Grant (P30 CA008748)
Footnotes
Conflict of interest:
None of the authors declare to have conflict of interest. No disclosures.
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