Cortisone (S3) - +60 Min.

Your body runs on a tightly choreographed daily rhythm of stress hormones. In the first hour after you wake, levels of cortisol (and its inactive partner, cortisone) climb sharply, then fall throughout the day. This +60 minute timepoint sits at the peak of that climb, and how high it rises tells you something about how your stress system is functioning that no random midday blood test can.

If you wake up exhausted, feel wired at night, or suspect chronic stress is wearing you down, a single cortisol number cannot answer the question. The morning surge can only be evaluated by sampling at multiple specific times. The S3 timepoint, taken 60 minutes after waking, is the one most closely tied to the body's daily stress reserve.

What This Test Actually Measures

Saliva contains the free, biologically active fraction of your stress hormones. Inside salivary glands, an enzyme called 11-beta-HSD2 (a tissue-level converter) turns active cortisol into inactive cortisone. Because this conversion is so efficient in saliva, salivary cortisone often gives a more stable read of your free hormone status than salivary cortisol itself, particularly in women on oral estrogens, where blood cortisol numbers can be misleading.

The S3 sample, collected 60 minutes after waking, captures the back end of what researchers call the cortisol awakening response, or CAR. The CAR is a brief, sharp rise in stress hormones over the first 30 to 60 minutes after you open your eyes. It is one of the most repeatable features of the body's daily hormonal pattern, and a flattened or exaggerated CAR has been linked to a range of stress-related conditions.

Why the Morning Surge Matters

Most large-population evidence for stress hormones and disease comes from studies of cortisol, not cortisone specifically. But because salivary cortisone tracks the same underlying biology as free cortisol (and is produced from it), the patterns are biologically connected.

Cardiovascular Disease

In the Whitehall II study of 4,047 working adults, people whose cortisol stayed flatter across the day (instead of dropping normally from morning to night) had a higher risk of dying from cardiovascular causes during follow-up. A separate German cohort, KORA-F3, tracked 1,090 adults and reached a similar conclusion: dysregulated daily cortisol patterns were associated with cardiovascular mortality, while greater natural variation across the day appeared protective. A Mendelian randomization analysis pooling cohort and genetic data also concluded that elevated morning cortisol is a likely causal risk factor for cardiovascular disease, suggesting that lowering cortisol exposure is worth investigating as a preventive strategy.

Long-term cortisone exposure has been linked to heart disease as well. In the Lifelines cohort of 6,341 adults, hair cortisone (which captures months of average exposure rather than a single morning's reading) was a significant predictor of future cardiovascular events, particularly in younger adults. These were different specimens than salivary cortisone, but the underlying signal, your cumulative stress hormone load, is the same.

What this means for you: a healthy morning surge that drops cleanly through the day is a feature, not a bug. A flat curve, with little difference between waking and bedtime, is the pattern most consistently tied to worse heart outcomes.

Metabolic Health and Insulin Resistance

The same enzyme system that produces cortisone also reactivates it back into cortisol inside fat, liver, and muscle. Studies in obese adults have shown increased local cortisol regeneration in fat tissue, which appears to drive features of metabolic syndrome including central weight gain, higher fasting insulin, and poorer glucose control. The exact relationship between salivary cortisone at +60 min and these metabolic shifts has not been pinned down, but the broader pattern of a disturbed cortisol-cortisone balance and insulin resistance is well-documented.

HPA Axis Dysfunction and Stress-Related Symptoms

The HPA axis (the hypothalamic-pituitary-adrenal axis, your body's main stress-control loop) shapes the morning surge captured by S3. When this loop is overstimulated by chronic stress, sleep disruption, or trauma, the awakening response can become blunted, exaggerated, or shifted in timing. Salivary measurements at multiple timepoints, including S3, are commonly used in research and integrative medicine to map these patterns rather than a single instantaneous reading.

Adrenal Insufficiency Screening

Salivary cortisone has a real, validated diagnostic role in screening for adrenal insufficiency, the condition in which the adrenal glands fail to produce enough cortisol. In one screening study, home waking salivary cortisone predicted adrenal insufficiency with strong diagnostic accuracy (an area under the curve close to 1.0, where 1.0 is perfect), and the great majority of patients with normal results truly had a healthy adrenal response. A larger study of 204 participants confirmed that salivary cortisone is especially useful for detecting adrenal insufficiency in women on oral estrogens, where blood cortisol can be falsely elevated.

Note that traditional adrenal insufficiency screening uses cortisone or cortisol drawn 60 minutes after a synthetic ACTH injection, which is a different protocol than the S3 +60 min after waking sample. The two share the time label but answer different clinical questions. The S3 sample looks at how your own body launches its morning hormone surge, not how it responds to a stimulating injection.

Reference Ranges

Salivary cortisone at S3 is a Tier 3 research and specialty marker. Standardized clinical cutpoints across labs do not exist, and assay methods vary. The DUTCH Plus and similar panels report results against laboratory-derived reference ranges built from healthy adult populations, and your trend across timepoints (S1 through S5) matters more than any single value. Compare your results within the same lab over time for the most meaningful trend.

What you and your clinician are really looking at is the shape of your daily curve. A typical healthy pattern shows S1 (waking) lower than S2 (+30 min) and S3 (+60 min), with both peaks falling sharply by S4 (dinner) and S5 (bedtime). A flat curve, an inverted curve (high at night, low in the morning), or a missing or exaggerated awakening peak each suggest different patterns of HPA axis function worth investigating.

Why One Reading Is Not Enough

Salivary stress hormones are highly variable from day to day. A poor night of sleep, an early-morning argument, an unexpected work email, or even hitting snooze can shift a single morning's reading. Treat your first test as a baseline, not a verdict.

If you are making meaningful changes (sleep schedule overhaul, stress reduction practice, supplementation, exercise program, addressing chronic illness), retest in 3 to 6 months to see whether your curve is moving in the right direction. After that, an annual recheck is reasonable for ongoing tracking. The pattern you build over years tells you more than any single morning.

What to Do With an Abnormal Result

An unusual S3 reading by itself does not name a diagnosis. It is one piece of a daily pattern. Look at it alongside your other timepoints (S1, S2, S4, S5), your DHEA-S level if measured on the same panel, and clinical context like sleep quality, mood, energy, and weight.

If results suggest a flat curve, an absent awakening response, or persistently elevated evening levels, the next sensible steps are to evaluate sleep (consider a sleep study if apnea is plausible), screen for chronic stress and mood conditions, and review medications that affect the HPA axis. If your morning samples are very low across the board with symptoms like fatigue, weight loss, dizziness, or low blood pressure, that pattern warrants endocrinology referral and proper adrenal insufficiency workup with an ACTH stimulation test, not just repeat saliva sampling.

When Results Can Be Misleading

• Sample timing errors: the S3 sample must be collected exactly 60 minutes after waking. Even a 15-minute drift can change where on the awakening response curve you are sampling.
• Eating, drinking, brushing teeth, or smoking before collection: any of these can contaminate the saliva and skew results. Most labs require nothing in the mouth for 30 to 60 minutes before sampling.
• Acute illness, surgery, or recent intense exercise: physical stress within the prior 24 to 72 hours can transiently shift the morning curve up or down without indicating a chronic problem. Retest in a calmer week.
• Glucocorticoid medications (oral, inhaled, topical, or injected): these directly suppress your body's cortisol and cortisone production while they are active in your system. The reading reflects the medication, not your underlying stress response.