Impact of Late Afternoon Testosterone Testing
Drawing testosterone in the late afternoon will result in falsely low values in younger men (under 45 years) and may lead to an incorrect diagnosis of hypogonadism, but this diurnal variation diminishes significantly with age, becoming negligible in men over 70 years.
Magnitude of Diurnal Variation by Age
The diurnal decline in testosterone is most pronounced in younger men and progressively diminishes with advancing age:
- Men aged 30-40 years: Testosterone levels measured at 4:00 PM are 20-25% lower than morning (8:00 AM) values 1
- Men aged 45-64 years: Free and bioavailable testosterone show significantly lower afternoon values, though total testosterone may not differ substantially 2
- Men aged 70 years: Only a 10% difference exists between morning and afternoon measurements 1
- Men aged ≥75 years: No significant differences in total, free, or bioavailable testosterone based on draw time 2
Clinical Consequences of Afternoon Testing
Risk of False-Positive Hypogonadism Diagnosis
In one study, 17 men who had at least one testosterone measurement below 300 ng/dL when drawn after noon actually had normal testosterone levels at all three morning visits 1. This included:
- 5 of 8 men aged 30-47 years (63%)
- 4 of 9 men aged 66-80 years (44%)
This demonstrates that afternoon testing can incorrectly classify men as hypogonadal, particularly younger individuals.
Minimal Impact on Other Hormones
The diurnal variation is specific to testosterone. Much lower levels of diurnal variation were found for dihydrotestosterone, SHBG, LH, FSH, and estradiol at all ages 1, meaning these hormones can be measured throughout the day without concern.
Physiologic Mechanism
The diurnal variation in testosterone is driven by LH pulsatility rather than changes in testosterone clearance 3. When gonadotropin secretion is suppressed experimentally, the diurnal testosterone rhythm disappears, confirming that the morning peak reflects increased testicular secretion in response to early-morning LH pulses 3.
Both young and middle-aged men display a significant circadian rhythm with a minimum fall of 43% in total testosterone from peak to nadir, with the acrophase (peak) occurring between 7:00-7:30 AM 4.
Guideline-Based Recommendations
Standard Practice
The American Diabetes Association recommends measuring testosterone with a morning serum testosterone level 5, and the American Urological Association requires total testosterone measurement on two separate early morning occasions (between 8 AM and 10 AM) 6.
Special Populations Where Timing May Be Less Critical
In men aged ≥45 years, early morning draw time may not be critical for capturing total testosterone concentrations; however, when measuring free or bioavailable testosterone, an early morning draw time may be preferable for men aged <75 years 2.
Practical Algorithm for Interpreting Afternoon Testosterone Values
If Testosterone Was Drawn in Late Afternoon:
For men <45 years with low afternoon testosterone:
For men 45-70 years with borderline-low afternoon testosterone (231-346 ng/dL):
For men >70 years with low afternoon testosterone:
Critical Pitfalls to Avoid
- Never diagnose hypogonadism based on a single afternoon testosterone measurement in men under 70 years 6, 1
- Do not assume that afternoon values are equivalent to morning values, even in older men—guidelines universally require morning testing 5, 6
- Do not use afternoon testing to monitor testosterone replacement therapy—the pharmacokinetic monitoring recommendations assume morning baseline values 7