What are the optimal times to collect salivary cortisol samples for screening or diagnostic purposes?

Optimal Times for Salivary Cortisol Collection

For screening Cushing's syndrome, collect late-night salivary cortisol at the patient's usual bedtime (not strictly at midnight), and for evaluating the cortisol awakening response, collect the first sample immediately upon awakening (within 0-15 minutes), followed by samples at 15 and 30 minutes post-awakening. 1, 2

Late-Night Salivary Cortisol (LNSC) for Cushing's Syndrome Screening

Collection timing should occur at the patient's usual bedtime rather than requiring patients to stay awake until 2300-2400 hours, as cortisol nadir is tightly entrained to sleep onset, not clock time. 1, 2, 3 This approach:

• Achieves diagnostic sensitivity of 92-100% and specificity of 93-100% for detecting Cushing's syndrome 1, 2
• Reflects the physiologic principle that patients with Cushing's syndrome lose their normal circadian nadir of cortisol secretion 2
• Requires at least 2-3 samples collected on different nights to account for variability and detect cyclic hypercortisolism 1, 2

Research confirms that bedtime sampling (2025-2400 hours) produces a lower upper limit of normal compared to strictly timed 2300-2400 hour sampling, making it equally valid and more practical for patients. 3

Morning Cortisol Awakening Response (CAR)

The first "waking" sample must be collected within 0-15 minutes of awakening to accurately capture the cortisol awakening response. 4, 5 Critical timing considerations include:

• Delays >15 minutes between awakening and first sample collection significantly elevate the waking cortisol value and flatten the subsequent rise, leading to misleading results 4, 5
• The cortisol awakening response peaks approximately 30 minutes after awakening, requiring samples at 0,15, and 30 minutes post-awakening 6, 7
• Self-reported wake time performs equally well to polysomnography for determining adherence to sampling procedures, so patients can reliably self-collect at home 5

The awakening cortisol response is a distinct phasic response to the sleep-wake transition, separate from the underlying circadian rhythm, making precise timing essential. 7

Single Time-Point Collections for Biomarker Studies

When diurnal variations are unknown or when standardization is needed:

• Collect between 9:00-11:00 AM as a standardized single time window until effects of collection timing can be verified 8
• This afternoon timing avoids the high cortisol awakening response and captures relatively stable biomarker levels 8
• Alternative recommendation suggests 8:00-10:00 AM collection to capture the physiologic peak of cortisol secretion 8, 1

Critical Pre-Collection Requirements

Patients must adhere to these restrictions to ensure accurate results:

• No food or drink for 30-60 minutes before collection to avoid contamination and pH alterations 1
• No teeth brushing or oral hygiene products for 1-2 hours prior to minimize blood contamination 8, 1
• No smoking, vaping, or nicotine products for 4 hours before sampling 8, 1
• No alcohol consumption for 12 hours preceding the test 8, 1
• Abstain from strenuous physical activity for 24-48 hours before collection, as exercise significantly increases salivary cortisol levels 8, 1
• Avoid cognitive stressors and neurocognitive assessments immediately before sampling, as mental stress elevates salivary cortisol 8

Common Pitfalls to Avoid

• Never use salivary cortisol testing in shift workers or patients with disrupted circadian rhythms, as their cortisol peaks occur at different times and yield unreliable results 8, 1
• Topical hydrocortisone preparations must be avoided before testing, as they contaminate samples and cause false elevations 1
• Document medications affecting cortisol binding globulin (oral estrogens, pregnancy), as these increase total cortisol levels 1
• Note any acute infections within the past 2 weeks, especially upper respiratory tract infections, as immune markers elevate cortisol; ideally reschedule testing 2 weeks later 8
• Sleep disturbances correlate with decreased morning awakening salivary cortisol in patients with insomnia, potentially affecting interpretation 8, 1

Sample Handling After Collection

• Collect samples at room temperature, then immediately place on ice to minimize proteolytic activity 8, 1
• Steroid hormones like cortisol are stable at ambient temperature for extended periods, unlike protein biomarkers 8, 1
• For late-night samples, patients should collect at bedtime and immediately refrigerate or freeze until transport to the laboratory 2