Collection Timing Matters for Urine Testing

Posted by Ben White on April 10, 2019

Urine is rapidly becoming the preferred medium for neurotransmitter testing to ensure objective neurobiological assessment. This is because urine is the primary route of peripherally-produced neurotransmitter elimination, and it is non-invasive and cost-effective. This article looks at how dried urine testing provides a superior advantage over standard liquid urine collection methods — and why collection timing is critical to getting accurate results.

Is a 24-Hour Collection Necessary?

The gold standard of neurotransmitter testing in urine has traditionally involved an inconvenient 24-hour collection of liquid urine into a jug. The awkwardness of this type of collection is further compounded by the fact that in certain circumstances during the day, it is nearly impossible to collect every last drop — and a patient may miss some collections altogether.

To circumvent these issues, dried urine can be used in place of the liquid sample, offering many advantages. Collecting urine on filter cards at 4 time points throughout the day is non-invasive, simple, straightforward and discreet — allowing the clinician and patient to get an accurate 24-hour clinical picture without the enormous hassle.

For the neurotransmitter test, ZRT recommends collecting urine 4 times throughout the day: the first void of the morning; the second void approximately 2 hours after waking; in the early evening around dinner time; and at bedtime. This method ensures both ease of obtaining the sample and stability during shipping and handling.

Our Neurotransmitter & Dried Urine Hormone Test uses exactly this 4-time-point dried urine collection method to measure dopamine, serotonin, norepinephrine, epinephrine, GABA and glutamate alongside urine hormones — with specialist interpretation included.

Understanding Diurnal Rhythms in Neurotransmitter Testing

Why a Single Morning Sample Is Not Enough

Every now and then, providers and patients ask whether the first or second morning collection alone can be used as a substitute for the 4-time-point collection. The short answer is no — with good reason.

Neurotransmitter levels do fluctuate dramatically during the day, with some neurotransmitters having very distinct circadian fingerprints.

Averaged throughout the day, neurotransmitter levels as determined by the 4-time-point collection are a stable measure of the individual’s neurotransmitter status. However, neurotransmitter levels fluctuate dramatically during the day, with some having very distinct circadian fingerprints.

The Diurnal Pattern of Epinephrine and Norepinephrine

Norepinephrine and epinephrine display specific diurnal rhythms — levels are low when we sleep and steadily increase over the course of the day to regulate blood pressure, heart rate and blood sugar, and adjust the body’s response to stressors accordingly.

Specifically, levels of norepinephrine and epinephrine are lowest in the first urine collection of the day, reflecting the body’s production of these catecholamine molecules during the night. Levels begin to increase towards mid-morning, peak in the afternoon, and decrease by bedtime — so a graph of the levels over a typical 24-hour period takes the shape of an inverted U. It is because of these diurnal fluctuations that one cannot reliably exchange the first or second morning samples for the 24-hour one without first developing reference ranges from samples collected at very specific time points.

The same principle applies to cortisol and cortisone. Our All Day Cortisol Test (4-Point LCMS) captures the full diurnal cortisol rhythm at four time points — providing a clinically meaningful picture of HPA axis function that a single morning sample simply cannot deliver.

The First and Second Morning Samples Are Different

It is precisely because of these diurnal fluctuations that laboratories testing urine collected at a single time point in the morning ought not to advise their patients to interchange the first and second morning urine samples. Without reference ranges developed from samples collected at specific time points to account for circadian rhythmicity, the clinical result will likely be grossly inaccurate.

ZRT Study: Epinephrine in First vs Second Morning Void

In a ZRT study, 30 healthy volunteers donated their first and second morning urine and the results were compared for epinephrine. Epinephrine levels in the second morning void, as a general rule, run higher than the first morning void. For some volunteers, the second morning urine showed dramatically higher levels of epinephrine than the first morning sample. This means that if a patient collects their first morning sample and the reference range was established based on second morning values, the patient’s result will fall into the “low” clinical range — yet that may not be a valid result for that individual.

Additional analysis shows that epinephrine values in the 1st and 2nd morning voids do not correlate, with an extremely low R² value of 0.0324 (if things correlate, the R² value ought to be closer to 1).

The Advantage of Dried Urine Testing

Dried urine represents a tremendous advantage over liquid 24-hour urine collections for both patients and clinicians. When testing is done right — with the correct 4-time-point collection — it gives not just a blurry snapshot of a patient’s neurotransmitter status, but instead a focused assessment that can pinpoint abnormalities in a diurnal rhythm as well as an accurate assessment of overall neurotransmitter production.

For those who also want to assess melatonin alongside cortisol and sleep hormones using the same dried urine method, our Sleep Hormone Test (UDH I) measures melatonin (MT6s), cortisol and cortisone across the day from a simple at-home dried urine collection — providing a comprehensive picture of the sleep-wake hormone rhythm.

Related Reading

Originally by Dr. Kate Placzek, ZRT Laboratory. Reproduced with permission. Last reviewed: May 2026.

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