Cortisol Awakening Response CAR Research

A deep dive into the biology, measurement, modulating factors, and emerging controversies surrounding one of neuroendocrinology's most studied biomarkers

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Table of Contents

1. What Is the Cortisol Awakening Response?
2. The Biology Behind CAR: HPA Axis and Circadian Rhythms
3. Normal Timing and Peak Window for CAR
4. How CAR Is Measured in Research and Clinical Practice
5. Factors That Influence CAR Magnitude
6. CAR as a Biomarker: Stress, Burnout, and Mental Health
7. The 2024–2025 Controversy: Is CAR Truly a Response to Waking?
8. CAR and Brain Network Reconfiguration: 2024 PNAS Findings
9. Blunted vs. Exaggerated CAR: Clinical Significance
10. CAR in Special Populations: Clinical vs. Healthy Adults
11. How to Collect CAR Samples Without Timing Errors
12. Future Directions in CAR Research
13. Frequently Asked Questions

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Introduction

Every morning, within the first hour of waking, your body orchestrates a hormonal surge that researchers have spent decades trying to understand. That surge—the Cortisol Awakening Response (CAR)—has been measured in thousands of studies, proposed as a window into hypothalamic-pituitary-adrenal (HPA) axis health, and linked to stress, burnout, depression, immune function, memory, and even brain network organization. Yet in 2024 and 2025, a wave of methodologically rigorous studies began asking a surprisingly fundamental question: Is the CAR actually caused by waking up at all?

This post synthesizes the full body of cortisol awakening response research, from its foundational definitions and measurement protocols to the most current controversies reshaping the field. Whether you are a researcher designing a cortisol awakening study, a clinician interpreting morning cortisol research, or a curious reader trying to understand what your body does in those first drowsy minutes after the alarm goes off, this guide is built for you.

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1. What Is the Cortisol Awakening Response?

The cortisol awakening response refers to the rapid, pronounced increase in salivary or serum cortisol concentrations observed in the first 30 to 60 minutes after a person wakes from sleep. Unlike the broader diurnal cortisol curve—which describes a gradual peak in the early morning followed by a slow decline across the day—the CAR was historically characterized as a discrete, awakening-triggered event superimposed on top of that circadian baseline.

A widely cited 2009 review formally defined the CAR as a distinct rise in cortisol immediately after waking, typically peaking 30 to 45 minutes after awakening, before returning toward pre-waking levels by approximately 60 minutes post-wake. This definition provided researchers with a concrete, measurable window and helped establish the CAR as one of the most reproducible neuroendocrine phenomena measurable non-invasively in free-living humans.

From a conceptual standpoint, CAR cortisol was distinguished from baseline morning cortisol in two important ways. First, the area under the curve with respect to increase (AUCi)—calculated from the waking sample through the 30-minute and 60-minute samples—was used to isolate the reactive component of the morning rise from the absolute cortisol level at waking. Second, researchers argued that the CAR represented a psychobiological preparation response, priming the body and brain for the demands of the coming day, rather than merely reflecting overnight cortisol accumulation.

These interpretive frameworks made CAR research enormously attractive as a translational tool. If CAR reflected psychological preparedness, then blunted CAR might signal burnout or exhaustion, while elevated CAR might reflect anticipatory stress. This interpretive promise drove an explosion of research across clinical psychology, occupational health, chronobiology, and psychiatry throughout the 2000s and 2010s. As we will see, however, the clarity of that original definition has been substantially complicated by more recent data.

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2. The Biology Behind CAR: HPA Axis and Circadian Rhythms

To understand why the cortisol morning peak exists at all, you need to understand the two overlapping systems that govern cortisol secretion: the HPA axis and the circadian clock.

The HPA Axis

The hypothalamic-pituitary-adrenal (HPA) axis is the body's primary stress-response hormonal system. In its standard feedback loop:

• The hypothalamus releases corticotropin-releasing hormone (CRH)
• CRH stimulates the anterior pituitary to release adrenocorticotropic hormone (ACTH)
• ACTH travels via the bloodstream to the adrenal cortex, which releases cortisol
• Rising cortisol feeds back negatively to both the hypothalamus and pituitary, dampening further release

CAR HPA research has long positioned the awakening response as a sensitive marker of HPA reactivity. Because the axis can be regulated by psychological states, chronic stress, sleep disruption, and inflammatory signaling, CAR was theorized to offer a real-world, ecologically valid snapshot of HPA tone—something that laboratory stressor paradigms could not easily replicate.

The Circadian System

Cortisol secretion follows a well-established 24-hour rhythm driven by the suprachiasmatic nucleus (SCN) in the hypothalamus—the brain's master clock. Cortisol begins rising in the second half of the night, reaches its cortisol waking peak in the morning, and declines across the afternoon and evening. This rhythm is entrained by light, feeding schedules, and social timing cues.

The circadian rise in cortisol precedes awakening in most individuals, meaning cortisol is already climbing before a person opens their eyes. This observation, long noted in chronobiology, has become central to the 2024–2025 controversy discussed later in this post.

Where CAR Fits

For most of its research history, the awakening response cortisol was conceptualized as sitting at the intersection of these two systems: a circadian-primed event that was amplified or triggered by the act of awakening. The CAR HPA relationship was interpreted as reflecting both the state of the axis (how reactive is it?) and the psychological appraisal of the coming day (what challenges does the person anticipate?). This dual-process model gave the CAR explanatory power across a wide range of psychological and physiological outcomes—a flexibility that, as critics have noted, may also have contributed to inconsistent findings across studies.

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3. Normal Timing and Peak Window for CAR

Establishing what counts as a normal CAR requires precise attention to timing. The standard sampling protocol—developed through iterative refinement across multiple cortisol awakening study designs—calls for saliva samples at:

• 0 minutes: immediately upon waking (within 1–2 minutes of eye opening)
• +15 minutes: 15 minutes after waking
• +30 minutes: 30 minutes after waking
• +45 minutes (some protocols): 45 minutes after waking
• +60 minutes: 60 minutes after waking

Based on the 2009 review and subsequent replication work, the cortisol morning peak in healthy adults typically occurs 30 to 45 minutes after awakening, reaching concentrations approximately 50 to 160% higher than the waking-moment sample. The magnitude varies substantially across individuals and days, which is part of why the AUCi metric—rather than a single peak value—became the preferred index in most morning cortisol research.

How Circadian Phase Affects the Peak Window

A landmark 2022 circadian-system study introduced an important nuance: the magnitude of the CAR is not fixed—it varies systematically depending on when awakening occurs relative to the internal circadian clock. Researchers found that the largest CAR occurred when participants awakened approximately three hours before their habitual wake time, suggesting that the circadian system actively modulates CAR magnitude. Awakening at the biological nadir of the cortisol rhythm produced the largest relative increases, while awakening at the expected habitual time—when the circadian cortisol slope was already steep—produced comparatively smaller incremental rises.

This finding has important methodological implications: studies that do not control for, or at least assess, circadian phase and habitual wake time may be introducing substantial noise into CAR estimates. It also adds mechanistic context to one of the more consistent findings in the literature—that earlier waking times are associated with larger CAR responses—likely because earlier natural wakers tend to wake closer to the biological nadir.

Day-to-Day Variability

Intraindividual variability in CAR is considerable. Test-retest reliability estimates across single days are modest (ICC values often in the 0.3–0.5 range), which is why most research protocols collect CAR across at least two, preferably three, weekdays and average the results. Single-day CAR samples are generally considered insufficient for drawing conclusions about an individual's HPA-axis trait characteristics.

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4. How CAR Is Measured in Research and Clinical Practice

The methodological choices made in CAR cortisol measurement profoundly affect the reliability and interpretability of findings. This section covers the main matrices and approaches used across research and applied settings.

Salivary Cortisol

Salivary cortisol remains the gold standard for free-living CAR measurement. Its advantages are numerous:

• Non-invasive: participants collect samples at home without venipuncture
• Reflects free cortisol: saliva contains only the biologically active, unbound fraction (~1–3% of total plasma cortisol)
• Ecologically valid: samples are collected in natural waking environments, not laboratories
• Compatible with self-administered protocols: participants use commercially available salivette or swab-based devices

Participants are typically instructed to collect samples without eating, brushing teeth, or drinking anything other than water in the period before sampling, and critically, to record the exact time of waking and each sample—a step that is more difficult to enforce than it sounds.

Blood (Serum/Plasma) Cortisol

Blood sampling provides total cortisol concentrations and allows simultaneous measurement of ACTH, providing a fuller picture of HPA-axis dynamics. However, venipuncture in the first minutes of waking is practically difficult in free-living conditions and the stress of the procedure itself can elevate cortisol, confounding CAR estimates. Blood-based morning cortisol research is therefore more common in controlled inpatient or laboratory settings.

In Vivo Microdialysis

Microdialysis represents the most temporally precise method, allowing continuous subcutaneous measurement of interstitial cortisol at near-minute resolution. While it is invasive and technically demanding—making it unsuitable for large-scale epidemiological work—it has been pivotal in the 2024–2025 debate about whether CAR represents a genuine post-waking acceleration in cortisol release.

Two key 2024–2025 studies used high-resolution data (from microdialysis and continuous blood sampling, respectively) to demonstrate that cortisol secretion rate did not significantly increase after awakening—a finding that fundamentally challenges the "response to waking" framing. We return to this in detail in Section 7.

Hair Cortisol

Hair cortisol concentrations (HCC) reflect cumulative cortisol exposure over weeks to months and are increasingly used in stress research. However, HCC cannot capture the dynamic, within-hour changes needed to assess CAR and are not considered relevant for CAR-specific measurement.

Key Analytical Indices

Two area-under-the-curve (AUC) metrics dominate the CAR biomarker literature:

• AUCi (area under the curve with respect to increase): reflects the magnitude of the rise from waking baseline; considered the CAR-specific index
• AUCg (area under the curve with respect to ground): reflects the total cortisol output in the morning window, including baseline levels

Most CAR researchers recommend reporting both, as they capture different aspects of morning cortisol dynamics and may have different relationships with psychological and health variables.

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5. Factors That Influence CAR Magnitude

One of the most consistent themes in cortisol awakening response research is the remarkable sensitivity of the CAR to a wide array of biological, psychological, behavioral, and environmental factors. Understanding these modulators is essential for both interpreting study findings and designing valid research protocols.

Stress and Anticipated Demands

A systematic review and meta-analysis examining associations between CAR and psychological states found positive correlations between CAR increase and both work-related stress and general life stress. When individuals anticipated a demanding workday, faced chronic occupational stressors, or reported higher general stress levels, their CAR AUCi values tended to be elevated. Conversely, the same review found negative correlations between CAR and fatigue, burnout, and exhaustion—a pattern consistent with the idea that prolonged, unremitting stress eventually depletes HPA-axis reactivity, producing a blunted awakening response.

A 2013 review of CAR moderators in healthy individuals confirmed that CAR magnitude positively correlated with anticipated workload or challenge and negative prior-day experiences. People going into a high-stakes day showed larger rises; people who had experienced distressing events the previous day also showed elevated CAR the following morning, possibly reflecting ongoing psychological processing of those events.

Morning Ambient Light

The 2013 review also identified morning ambient light as a positive correlate of CAR magnitude. Light is a primary entraining signal for the circadian system, and brighter morning light exposure—particularly in the blue-wavelength range—activates the SCN and accelerates the shift toward waking physiology. This provides one plausible mechanism linking light conditions to the amplitude of the cortisol morning peak.

Practically, this means that studies collecting CAR across different seasons, geographic latitudes, or in participants who wake in varying light conditions may need to account for ambient light as a covariate.

Wake Time and Circadian Alignment

As described in Section 3, the endogenous circadian system modulates CAR magnitude based on the phase relationship between awakening and the internal clock. Earlier natural wake times—associated with a "morning chronotype"—tend to produce larger CARs, while later chronotypes waking at their habitual late times may show comparatively attenuated responses.

The 2022 circadian study's finding that awakening three hours before habitual wake time produced the largest CAR further underscores that it is the circadian context of waking, not simply the act of waking, that shapes the response.

Menstrual Cycle Phase

Female sex is a well-documented source of CAR variability. The 2013 review noted that CAR magnitude was positively associated with the ovulatory period in females—likely reflecting interactions between estrogen, progesterone, and HPA-axis reactivity. Researchers studying CAR in female participants are therefore advised to either control for menstrual cycle phase or to stratify analyses accordingly.

Sleep Quality and Duration

Poor sleep quality—including frequent awakenings, reduced slow-wave sleep, and shorter sleep duration—has been associated with alterations in morning cortisol dynamics. Sleep fragmentation may blunt the sharp morning rise, though findings across studies are not entirely consistent, partly due to variation in how sleep quality is measured and whether polysomnography or self-report is used.

Age and Sex

CAR magnitude and timing vary across the lifespan. Adolescents tend to show larger CARs than older adults, partly reflecting HPA-axis maturation and partly reflecting the pronounced sleep timing shifts associated with puberty. In aging, HPA regulation becomes less precise, and the diurnal cortisol slope tends to flatten—though whether CAR per se is attenuated in healthy aging remains somewhat contested.

Men and women show differences in both absolute cortisol levels and CAR magnitude, with hormonal fluctuations in women adding variability that is rarely captured in studies that treat sex as a binary covariate without accounting for reproductive status.

Physical Exercise

Acute morning exercise before or during the CAR sampling window is a significant confound, as exercise is itself a potent HPA activator. Standard CAR protocols instruct participants to remain sedentary during the sampling window. Studies examining CAR in athletes or active populations must carefully distinguish between exercise-induced cortisol responses and the CAR itself.

Medications and Substances

Glucocorticoid medications (including inhaled and topical formulations), oral contraceptives, antidepressants (particularly SSRIs), alcohol, caffeine, and nicotine all influence HPA-axis dynamics to varying degrees. Many cortisol awakening study designs explicitly exclude participants on these substances or treat medication status as a covariate.

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6. CAR as a Biomarker: Stress, Burnout, and Mental Health

The promise of CAR as a CAR biomarker rests on the hypothesis that it indexes something biologically meaningful about HPA-axis tone—something stable enough to reflect a person's chronic stress exposure, vulnerability to mental health conditions, or current psychological state. Decades of research have produced a complex, nuanced picture.

Work Stress and Burnout

The meta-analytic evidence linking CAR cortisol to occupational stress is among the most replicated in the field. The systematic review and meta-analysis referenced earlier found that elevated CAR was consistently associated with higher perceived work demands and greater general life stress, while blunted CAR was associated with burnout, exhaustion, and fatigue. This bidirectional pattern—elevated in early or acute stress, blunted in chronic or depleted states—has been proposed as a marker of HPA-axis allostatic load: early stress mobilization followed by eventual dysregulation.

In occupational health settings, this has made CAR an attractive candidate biomarker for identifying individuals at risk of burnout before they reach clinical thresholds. However, the effect sizes reported in the meta-analytic literature are generally modest, and the overlap between CAR distributions in stressed versus non-stressed groups is substantial—limiting its diagnostic precision at the individual level.

Depression and Anxiety

The relationship between CAR and depression is complex and appears to depend on depression subtype, severity, and whether depression is current or in remission. Some studies report elevated CAR in melancholic depression, consistent with the hyperactivity of the HPA axis documented in this subtype. Others report blunted CAR in atypical depression or chronic, treatment-resistant cases. Anxiety disorders show similarly heterogeneous patterns, with post-traumatic stress disorder (PTSD) often associated with a blunted CAR—particularly in more severe or chronic presentations.

Immune Function and Inflammation

Cortisol is a potent immunomodulator, and the morning cortisol surge has long been hypothesized to prime immune cells for the challenges of the day. CAR has been associated with natural killer cell activity, pro-inflammatory cytokine levels, and inflammatory biomarkers in several studies. Whether CAR-linked immune effects are clinically significant in healthy adults remains an open question, though the findings are mechanistically plausible.

Cognitive Function and Memory

The glucocorticoid system plays a well-documented role in memory consolidation and executive function. Morning cortisol research has found associations between higher morning cortisol (including CAR) and declarative memory performance, with inverted-U relationships suggesting that moderate cortisol levels optimize cognitive function while very high or very low levels impair it. We return to the brain network implications of this in Section 8.

Limitations as a Biomarker

Despite its promise, a 2025 major review in Endocrine Reviews and related commentary highlight persistent limitations of the CAR as a clinical biomarker:

• Low single-session reliability: a single morning's CAR is a poor estimate of trait-level HPA function
• Confounding by adherence: if participants do not collect samples at the exact required times, the AUCi calculation becomes unreliable
• Heterogeneous protocols: the lack of standardization across studies makes meta-analytic pooling difficult
• Ambiguity of mechanism: if, as recent evidence suggests, the CAR partly reflects pre-waking circadian cortisol dynamics rather than a waking-triggered response, its interpretation as an index of awakening-specific HPA reactivity needs revision

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7. The 2024–2025 Controversy: Is CAR Truly a Response to Waking?

The most intellectually consequential development in CAR research in the past two years is a series of studies that have directly challenged the foundational assumption that the cortisol rise after waking is caused by the act of waking.

The 2024 Proceedings of the Royal Society B Study

In a carefully designed 2024 study published in Proceedings of the Royal Society B, investigators studied 201 healthy volunteers and directly compared cortisol secretion dynamics in the hour before versus the hour after awakening. Their finding was striking: there was no evidence that cortisol secretion rate increased as a result of awakening. Cortisol was already rising before waking, and the rate of change in cortisol concentrations was not significantly different between the pre- and post-waking periods.

This study used high-resolution sampling that allowed the researchers to separate the continuous circadian rise from any hypothetical awakening-triggered acceleration. The data suggested that what we observe as the "awakening response" may simply be the continuation of a cortisol rise that began well before the alarm clock—making the act of waking incidental rather than causal.

The 2025 Brain Neuroscience Advances Review

A 2025 review published in Brain Neuroscience Advances titled "The cortisol awakening response: Fact or fiction?" synthesized these newer data and reached a provocative conclusion: waking per se is not accompanied by a distinct acceleration in cortisol release. Drawing on the 2024 Royal Society B findings and associated in vivo microdialysis data, the review argued that the best predictor of increased cortisol release around the time of waking was actually the cortisol level in the hour before awakening—not the act of waking itself.

Put differently: a steeply rising pre-waking cortisol trajectory predicts a high post-waking cortisol level, and calling the post-waking portion a "response to awakening" may be an artifact of our traditional sampling approach rather than a reflection of a biological event triggered by waking.

The 2025 European Journal of Applied Physiology Replication

The 2025 Brain Neuroscience Advances review also cites a 2025 replication and extension study published in the European Journal of Applied Physiology under the title "Is the cortisol awakening response truly a response to awakening?", which used overnight continuous sampling to replicate and extend the Royal Society B findings. The convergence of these independent datasets significantly strengthens the case for revisiting the standard CAR framework.

The 2025 Endocrine Reviews Major Review

The 2025 Endocrine Reviews publication on "The Cortisol Awakening Response: Regulation and Functional Significance" provides the field's most comprehensive current synthesis, acknowledging these challenges while also reviewing the extensive evidence for CAR's associations with psychological and health outcomes. This review is likely to serve as the reference standard for the field for the coming decade, and it stops short of dismissing the CAR as scientifically meaningless—instead calling for more precise mechanistic models that account for both circadian and awakening-specific contributions.

What This Means for CAR Interpretation

The controversy does not render prior cortisol awakening response research invalid. The associations between CAR (as traditionally measured) and stress, burnout, brain function, and health outcomes remain real and replicable. What changes is the mechanism we invoke to explain them. If the pre-waking cortisol trajectory is the primary driver, then CAR AUCi may be better understood as an index of the steepness and height of the early-morning circadian cortisol slope rather than a response to awakening per se.

For morning cortisol research design, this implies that collecting a sample immediately before waking—while technically difficult in free-living settings—may provide more mechanistic information than the traditional post-wake sampling window alone.

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8. CAR and Brain Network Reconfiguration: 2024 PNAS Findings

While the mechanistic origin of CAR was being debated in chronobiology and endocrinology journals, neuroscience was adding a new dimension to the story. A 2024 study published in Proceedings of the National Academy of Sciences (PNAS) reported that the cortisol awakening response prompts dynamic reconfiguration of brain networks involved in emotional and executive functioning.

What the Study Found

Using resting-state fMRI paired with salivary CAR measurement, the investigators tracked how functional brain network organization shifted across the morning wake-up period. They found that larger CARs were associated with more pronounced changes in network connectivity—specifically in regions associated with:

• Emotional regulation: amygdala-prefrontal connectivity patterns shifted in ways consistent with heightened emotional salience processing
• Executive function: networks supporting working memory, cognitive control, and flexible attention showed reorganization that tracked with the CAR magnitude

Interpreting the Functional Significance

This finding provides a compelling mechanistic bridge between the hormonal event (or circadian-phase event, given the ongoing debate) and the psychological functions that CAR has long been theorized to support. If cortisol—regardless of whether it is triggered by waking or already rising pre-wake—reorganizes brain networks toward greater emotional and executive readiness in the morning hours, then its functional significance is real even if its mechanistic origin needs reframing.

The PNAS findings are also consistent with earlier behavioral data showing that higher morning cortisol correlates with better declarative memory and more efficient cognitive performance in the early daytime window. Cortisol's role as a neuromodulator in prefrontal and limbic circuits provides the mechanistic substrate for these effects.

Implications for the CAR Biomarker Framework

For researchers using CAR cortisol as a biomarker, the PNAS data suggest that what matters functionally is less whether the rise was triggered by waking and more whether the rise occurred and how large it was. A blunted morning cortisol rise—however caused—may translate into suboptimal brain network priming, with downstream consequences for emotional regulation and cognitive performance across the day.

This reframes the clinical significance of a blunted CAR: rather than specifically indicating "impaired awakening response," it may indicate inadequate circadian-phase cortisol mobilization with functional neurobiological consequences.

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9. Blunted vs. Exaggerated CAR: Clinical Significance

In the clinical and research literature, two patterns of CAR abnormality have received the most attention: a blunted CAR (smaller-than-expected rise) and an exaggerated CAR (larger-than-expected rise). Understanding what these patterns may mean—while acknowledging the substantial overlap between clinical and healthy populations—is important for applying CAR biomarker science in real-world contexts.

Blunted CAR

A blunted awakening response cortisol pattern—characterized by a flat or minimal rise from the waking sample to the 30-minute peak—has been associated with:

• Burnout and exhaustion: The meta-analytic evidence consistently links blunted CAR with occupational burnout, consistent with a hypocortisolism phenotype in which the HPA axis has become downregulated following prolonged stress exposure
• PTSD: Multiple studies in trauma populations report blunted CAR, particularly in individuals with more severe or chronic PTSD. HPA-axis hyporeactivity in PTSD may reflect enhanced negative feedback sensitivity (supported by the low-dose dexamethasone suppression findings in PTSD) that limits morning cortisol mobilization
• Atypical depression: In contrast to melancholic depression, atypical depression is characterized by HPA hyporeactivity, and blunted CAR has been reported in some atypical depression samples
• Chronic fatigue syndrome: Studies in CFS/ME populations have reported blunted CAR in at least a subset of patients, consistent with broader HPA-axis hyporeactivity in this condition
• Adverse childhood experiences: Early life adversity has been associated with blunted CAR in adulthood, potentially reflecting long-term HPA programming effects

Exaggerated CAR

A larger-than-average cortisol waking peak has been associated with:

• Acute and anticipatory stress: As described, higher CAR is found in individuals facing demanding upcoming days or general life stress
• Melancholic depression: HPA hyperactivity is a well-documented feature of melancholic depression, and elevated morning cortisol is part of the dexamethasone non-suppression pattern characteristic of this subtype
• First-episode psychosis: Some studies report elevated CAR in early psychosis, consistent with broader HPA-axis hyperactivation in early-stage psychotic disorders
• Chronic stress in younger adults: In contrast to the blunting seen with long-term burnout, moderate chronic stress in younger, otherwise healthy adults may still manifest as elevated CAR

The Inverted-U Problem

The clinical literature's most consistent challenge is that both elevated and blunted CAR can be pathologically relevant—just in different conditions and time courses. This means that a single CAR value cannot be interpreted without context. An elevated CAR may represent appropriate stress mobilization in a healthy person facing real demands, or it may reflect early-stage dysregulation. A blunted CAR may represent healthy low-stress states or pathological HPA exhaustion.

Contextual information—including chronic stress history, psychological symptoms, sleep quality, medication use, and chronotype—is essential for meaningful CAR interpretation.

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10. CAR in Special Populations: Clinical vs. Healthy Adults

Morning cortisol research has now accumulated findings across a remarkably diverse range of populations. Here we summarize key patterns in major clinical and non-clinical groups.

Healthy Adults

In healthy, non-clinical adult populations, CAR cortisol shows the expected 30–45-minute peak pattern, with substantial inter- and intraindividual variability. CAR is larger on weekdays than weekends (possibly reflecting anticipatory stress of work demands), larger after nights of poorer sleep, and modulated by the light, chronotype, and circadian factors discussed earlier. Importantly, the 2024–2025 studies challenging the mechanistic basis of CAR were conducted primarily in healthy volunteer samples, reminding us that even in the absence of psychopathology, the mechanisms underlying CAR deserve continued scrutiny.

Shift Workers

Shift workers present a natural experiment in circadian disruption. Their HPA-axis dynamics are profoundly altered by misalignment between the endogenous circadian clock and the external schedule of waking and sleep. CAR in shift workers is generally more variable and often blunted relative to day workers, consistent with the known HPA-axis effects of chronic circadian misalignment. The 2022 finding that circadian phase modulates CAR magnitude is particularly relevant here—shift workers may experience their post-sleep awakening at atypical circadian phases, producing CAR patterns that differ fundamentally from day-working reference populations.

Adolescents

Adolescents show larger absolute CAR responses than most adult populations, and their later chronotype—driven by pubertal hormonal changes and school scheduling—means that many teenagers are awakening at a biologically unfavorable circadian phase on school days. Some researchers have proposed that the mismatch between school start times and adolescent chronotype produces chronically disrupted CAR patterns that may affect mood, cognitive performance, and stress reactivity in this age group.

Older Adults

In healthy aging, HPA-axis regulation becomes less precise. The circadian cortisol rhythm tends to flatten, with a less pronounced morning peak and higher evening nadir. CAR-specific changes in aging are less well-characterized than the broader diurnal changes, but studies generally report smaller CARs in older adults, consistent with HPA system aging.

Children

CAR research in children has expanded considerably in the past decade, driven partly by interest in how early adversity programs the HPA axis. Children exposed to maltreatment, poverty, or parental mental illness show altered CAR patterns, but the direction of effects is inconsistent across studies—possibly because developmental stage, type of adversity, and concurrent resilience factors all modulate outcomes differently.

Clinical Psychiatric and Medical Populations

Beyond the conditions discussed in Section 9, CAR has been studied in populations including:

• Type 2 diabetes and metabolic syndrome: Elevated morning cortisol and altered CAR dynamics have been reported, reflecting HPA-metabolic axis interactions
• Chronic pain conditions: Some fibromyalgia and chronic back pain studies report blunted CAR, consistent with central sensitization and HPA hyporeactivity patterns
• Cancer patients: Cancer and its treatment (particularly chemotherapy) disrupt circadian cortisol rhythms, including the morning peak
• Cardiovascular disease: The morning cortisol peak has been associated with increased platelet aggregation and cardiovascular risk, making CAR dynamics potentially relevant to morning cardiovascular event clustering

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11. How to Collect CAR Samples Without Timing Errors

The validity of any cortisol awakening study depends enormously on protocol adherence. Because the CAR window spans only 30–60 minutes, even small timing errors can produce large artifacts in AUCi calculations. This section covers best practices based on the current literature.

The Compliance Problem

Studies comparing self-reported sample times with objective timestamps embedded in electronic sampling devices have repeatedly found that participants systematically misreport their waking times—typically by underreporting the time between waking and the first sample. This creates an artificial inflation of the AUCi because the "baseline" sample is actually collected several minutes after waking, when cortisol has already started to rise.

The magnitude of this artifact can be substantial. Estimates suggest that a 10-minute delay in the first sample (with the participant reporting it as taken immediately upon waking) can artifactually inflate AUCi by a clinically significant margin.

Objective Timestamping

The gold standard for morning cortisol research compliance verification is the use of electronic monitoring devices—either dedicated sampling monitors (e.g., MEMS-cap devices on saliva tubes) or smartphone-based apps that timestamp and optionally photograph samples. Studies using objective monitoring consistently find lower apparent CAR values than those relying on self-report alone, reflecting removal of the compliance artifact.

Actigraphy—wrist-worn accelerometry—can additionally verify wake time and sleep timing, allowing researchers to confirm that participants' self-reported wake times align with objective movement data.

Practical Instructions for Participants

Standard instructions for CAR sample collection should include:

1. Collect the first sample within 1–2 minutes of waking, before getting out of bed if possible, before any food or drink, and before brushing teeth
2. Do not set a secondary alarm: alarm-based awakening may be acceptable, but participants should be aware that the sample must be collected immediately
3. Record the exact time on the sample tube and in a diary, or use an electronic app
4. Remain sedentary during the sampling window: no exercise, major activity, or showering until after the 60-minute sample
5. Avoid eating, drinking (other than plain water), smoking, or chewing gum during the sampling window
6. Collect on workdays if the research question concerns work-related CAR: weekend CAR is systematically lower in most employed adults and should not be mixed with workday samples without stratification
7. Collect on at least 2–3 consecutive days: averaging across days substantially improves the reliability of the CAR estimate

Handling and Storage

Saliva samples should be kept cool (refrigerated if possible) after collection and frozen at −20°C or below until laboratory assay. Repeated freeze-thaw cycles degrade salivary cortisol and should be avoided. Most commercially available enzyme-immunoassay (EIA) and luminescence immunoassay (LIA) kits are validated for salivary cortisol and provide acceptable sensitivity for the concentrations found in the CAR window.

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12. Future Directions in CAR Research

The field of cortisol awakening response research stands at an unusually dynamic juncture. The convergence of high-resolution continuous sampling technologies, large-scale neuroimaging studies, and methodological challenges to foundational assumptions is creating pressure for significant conceptual and practical revision. Here are the directions that are most likely to shape the field in the coming years.

Mechanistic Clarification: Circadian vs. Awakening-Triggered Components

The most urgent scientific priority is to definitively characterize the relative contributions of the endogenous circadian cortisol rise and any awakening-triggered component. This will require:

• Large studies combining polysomnography with high-frequency sampling (ideally microdialysis or continuous blood sampling) across manipulated sleep timing conditions
• Studies in shift workers, jet-lag protocols, and forced desynchrony paradigms, where circadian phase and wake time can be experimentally decoupled
• Mathematical modeling approaches that can partition circadian and ultradian cortisol pulsatility from any hypothetical awakening signal

Standardization of Protocols

The field would benefit substantially from consensus guidelines on sampling protocol, compliance verification, and analytical methods. An expert panel recommendation similar to those developed for other biomarker areas would help reduce heterogeneity and improve cross-study comparability.

Integration with Wearable Technology

As consumer-grade wearables become increasingly capable of detecting physiological signals—including optical heart rate, skin conductance, and sleep-wake transitions—there is growing potential for integrating actigraphy, photoplethysmography-based sleep staging, and even cortisol patch technologies (currently in development) into morning cortisol research. These tools could dramatically improve compliance monitoring and allow CAR data collection at unprecedented scales.

CAR in Longitudinal and Intervention Studies

Most CAR cortisol research has been cross-sectional. Longitudinal studies tracking CAR across months or years—particularly in relation to evolving stress exposures, psychiatric trajectories, or aging—are needed to establish the predictive validity and clinical utility of CAR as a trait-level biomarker. Similarly, intervention studies examining whether treatments that normalize HPA-axis function (cognitive-behavioral therapy, stress management programs, exercise interventions, sleep optimization) produce detectable changes in CAR would significantly advance its clinical relevance.

Personalized Medicine Applications

If CAR can be reliably measured in real-world settings using validated electronic protocols, and if normative data can be established across age, sex, and chronotype groups, it may eventually be possible to use CAR as one component of personalized stress biology profiles—helping identify individuals at elevated risk for HPA-related health outcomes and tailoring interventions accordingly.

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13. Frequently Asked Questions

Q: What is the cortisol awakening response (CAR)? A: The cortisol awakening response is the rapid increase in salivary or serum cortisol concentrations observed in the 30–45 minutes after waking from sleep. It has been studied as an index of HPA-axis function, psychological stress, and morning preparedness. Recent research has questioned whether this rise is specifically triggered by the act of waking or whether it primarily reflects the continuation of the circadian cortisol rise that begins before awakening.

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Q: Is CAR a reliable biomarker of HPA-axis function? A: CAR shows moderate test-retest reliability across days and is meaningfully associated with stress, burnout, depression subtypes, and brain function at the group level. However, single-session reliability is low, and substantial overlap between clinical and healthy populations limits its diagnostic utility at the individual level. The 2024–2025 mechanistic challenges add further nuance: CAR may reflect circadian cortisol slope steepness as much as awakening-specific HPA reactivity.

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Q: What is the normal timing and peak window for CAR? A: In healthy adults, cortisol typically peaks 30–45 minutes after awakening, rising approximately 50–160% from the waking-moment baseline. The magnitude is modulated by circadian phase, stress levels, light exposure, and chronotype.

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Q: How is CAR measured in practice? A: Salivary cortisol is the standard method, collected at waking (0 minutes), +15, +30, and +60 minutes post-wake. The area under the curve with respect to increase (AUCi) is the preferred index. Electronic timestamping is recommended to verify compliance, as self-reported timing is frequently inaccurate.

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Q: What factors influence CAR? A: CAR magnitude is positively associated with morning ambient light, earlier wake times, anticipated workload, prior-day stress, and the ovulatory phase in females. It is negatively associated with burnout, fatigue, and exhaustion. Circadian phase at waking is a major modulator, with awakening about three hours before habitual wake time producing the largest response.

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Q: Is CAR a response to waking itself, or part of the circadian cortisol rhythm? A: This is now actively debated. A 2024 study of 201 healthy volunteers found no evidence that cortisol secretion rate increased following awakening; cortisol was already rising before waking, and the rate of change was not different pre- versus post-wake. A 2025 review concluded that waking per se is not accompanied by a distinct acceleration in cortisol release, and that the best predictor of post-waking cortisol levels is the cortisol level in the hour before awakening.

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Q: What does a blunted vs. exaggerated CAR mean clinically? A: A blunted CAR is associated with burnout, PTSD, chronic fatigue, and atypical depression—suggesting HPA hypofunction. An exaggerated CAR is associated with acute/anticipatory stress and melancholic depression—suggesting HPA hyperactivation. Context is essential, as both patterns can occur in healthy individuals under different circumstances.

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Q: Can CAR predict stress, burnout, depression, anxiety, or fatigue? A: At the group level, yes—meta-analytic evidence confirms associations between CAR and these outcomes. However, effect sizes are modest and individual-level prediction is limited. CAR is best used as one component of a broader assessment rather than as a standalone diagnostic biomarker.

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Q: How should CAR samples be collected to avoid timing errors? A: The first sample must be collected within 1–2 minutes of waking, before eating, drinking, or brushing teeth. Samples should be collected across at least 2–3 days and averaged. Electronic timestamping devices or apps should be used to verify compliance. Participants should remain sedentary and avoid food, drink, and smoking during the sampling window.

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Q: How do CAR findings differ between healthy adults and clinical populations? A: Healthy adults show reliable CAR patterns modulated by stress and chronotype. Clinical populations tend to show more pronounced deviations—blunted CAR in PTSD, burnout, and atypical depression; elevated CAR in melancholic depression and early psychosis—though overlap is substantial and findings across studies are not fully consistent.

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Conclusion

The Cortisol Awakening Response CAR Research landscape has evolved dramatically since the 2009 review that gave the field its operational definition. What began as a straightforward story—a cortisol peak triggered by waking, reflecting HPA-axis readiness for the day—has become something considerably more complex and, arguably, considerably more interesting.

The 2024 Proceedings of the Royal Society B study of 201 healthy volunteers, the 2025 Brain Neuroscience Advances review, the 2025 Endocrine Reviews synthesis, and the European Journal of Applied Physiology replication study together represent a paradigm-shifting challenge to the foundational assumption that waking causes the morning cortisol surge. If cortisol was already rising before participants opened their eyes—and if the best predictor of post-waking levels is the pre-waking cortisol trajectory—then what we call the "awakening response" may be better understood as the morning circadian cortisol peak, measured through the convenient lens of waking rather than triggered by it.

Yet this mechanistic revision does not diminish the functional importance of the cortisol morning peak. The 2024 PNAS findings that CAR prompts dynamic reconfiguration of brain networks in emotional and executive functioning confirm that whatever drives the morning cortisol surge, its neurobiological consequences are real and consequential. The meta-analytic associations between CAR biomarker patterns and stress, burnout, mental health, and cognitive function remain the empirical foundation of the field—they require reinterpretation in light of new mechanistic data, not abandonment.

For researchers, the priority now is clear: high-resolution continuous sampling studies, longitudinal designs, and consensus protocols that can resolve the mechanistic questions while preserving the translational promise of morning cortisol as a window into human stress biology. For clinicians and practitioners, CAR remains a useful—if imperfect and context-dependent—indicator of HPA-axis dynamics, best interpreted alongside clinical history, psychological assessment, and other biomarkers.

The story of CAR cortisol is, in the best scientific tradition, a story that keeps revising itself. The next chapter, being written right now in endocrinology, chronobiology, and cognitive neuroscience laboratories around the world, promises to be the most scientifically rigorous yet.

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