At what time is a salivary hormone collection for cortisol typically recommended?

Timing of Salivary Cortisol Collection

Salivary cortisol should be collected at late-night/bedtime (typically between 10:00 PM and midnight) for screening Cushing's syndrome, or in the early morning immediately upon awakening for assessing the cortisol awakening response and diurnal patterns. 1, 2

Late-Night Collection for Cushing's Syndrome Screening

The most clinically important timing for salivary cortisol is late-night collection at the patient's usual bedtime, which demonstrates 92-100% sensitivity and 93-100% specificity for detecting Cushing's syndrome. 1, 2

Specific Timing Recommendations:

• Collection should occur at the patient's usual bedtime rather than strictly at midnight, as the cortisol nadir is tightly entrained to sleep onset 1, 2
• Acceptable collection window is between 10:00 PM (2200 hours) and midnight (2400 hours) 3
• Research confirms no significant difference between samples collected at 10:00 PM versus 11:00 PM 4
• At least 2-3 samples should be collected on different nights to account for variability and detect cyclic hypercortisolism 1, 2

Physiological Rationale:

The late-night timing capitalizes on the loss of normal circadian nadir of cortisol secretion that occurs in Cushing's syndrome, making this the most diagnostically valuable collection time 1

Morning Collection for Diurnal Pattern Assessment

For evaluating diurnal cortisol patterns and the cortisol awakening response, collection should occur immediately upon awakening (typically 8:00-9:00 AM), as this captures the physiologic peak of cortisol secretion. 2, 5

Morning Collection Advantages:

• Morning samples (immediately after awakening) show the smallest day-to-day variation (mean coefficient of variation: 43.1%) compared to evening samples (100.3%) 5
• From a reproducibility standpoint, collecting saliva immediately after awakening is the most advantageous time for evaluating cortisol concentration 5

Standardized Single Time-Point Collection for Research

When diurnal variations are unknown or being controlled for (such as in Alzheimer's disease biomarker research), a standardized collection window of 9:00-11:00 AM is recommended to minimize circadian effects. 6

This mid-morning timing avoids the high cortisol awakening response while maintaining relatively stable biomarker levels 6

Critical Pre-Collection Requirements (Regardless of Timing)

Mandatory Restrictions Before Collection:

• No food or drink (except plain water) for at least 1 hour before collection 6, 2
• No smoking, vaping, or nicotine products for 4 hours before sampling 6, 2
• No alcohol consumption for 12 hours before testing 6, 2
• No teeth brushing or oral hygiene products for 1-2 hours before collection 6, 2
• No strenuous physical activity for 24-48 hours before collection, as exercise significantly elevates salivary cortisol 6, 7, 2

Additional Considerations:

• Mouth should be rinsed with water 10 minutes before collection 6
• Patient should rest for 10 minutes before collection to minimize stress-related cortisol elevation 6
• Cognitive assessments should be performed AFTER saliva collection to avoid stress-induced cortisol elevation 6

Common Pitfalls to Avoid

• Do not require patients to stay awake until a strict midnight collection time; allow collection at their usual bedtime 3
• Avoid topical hydrocortisone preparations before testing, as they contaminate samples 2
• Be aware that single bedtime samples may have higher day-to-day variability (100% coefficient of variation) compared to morning samples (43%) 5
• Patient compliance with timed collections is often poor in home settings, so electronic monitoring or assisted collection may improve accuracy 8, 9