CONTEXT:The hypothalamic-pituitary-adrenal (HPA) axis is the key homeostatic system regulating the response to surgical stress. Imbalances in HPA axis hormones increase morbidity and mortality in children after cardiac surgery. Despite this, the physiology of the HPA axis in children undergoing cardiac surgery is poorly understood, leading to controversies in clinical practice. OBJECTIVE:To characterise dynamic HPA axis responses in children undergoing cardiac surgery and to determine age- and procedure-related differences in cortisol and cortisone physiology. METHODS:We recruited children (0-18 years) undergoing cardiac surgery with cardiopulmonary bypass or cardiac catheterisation. Tissue-free cortisol and cortisone were sampled every 20 minutes for up to 24 hours via microdialysis, alongside serum adrenocorticotropic hormone (ACTH), cortisol, cortisol-binding globulin (CBG), and inflammatory markers. We developed dynamic markers to quantify age- and procedure-dependent differences in hormonal responses and built a mathematical model to explain them. RESULTS:Neonates undergoing surgery showed higher free cortisol and cortisone AUC and peak concentrations than catheterisation patients. Neonates had higher peaks of cortisol and cortisone than older children undergoing surgery. The much higher tissue cortisone levels observed in neonates can be explained by enzymatic interconversion between cortisol and cortisone, likely due to persistent foetal high 11-βHSD2 activity and reduced 11-βHSD1 activity.Low post-operative blood cortisol and CBG values in neonates resulted in high free cortisol peaks in interstitial fluid during and after surgery. CONCLUSION:Neonates differ physiologically, with higher free cortisol levels that more readily diffuse into interstitial tissues, with implications for perioperative management.

People with severe mental illness have high rates of obesity, type 2 diabetes, and cardiovascular disease. Emerging evidence suggests that metabolic dysfunction may be causally linked to the risk of severe mental illness. However, more research is needed to identify reliable metabolic markers which may have an impact on mental health outcomes, and to determine the mechanisms behind their impact. In the METPSY research study, we will investigate the relationship between metabolic markers and clinical outcomes of severe mental illness in young adults. We will recruit 120 young adults aged 16–25 years living in Scotland with major depressive disorder, bipolar disorder, schizophrenia, or no severe mental illness (controls) for a prospective observational study. We will assess clinical symptoms at three in-person visits (baseline, 6 months, and 12 months) using the Structured Clinical Interview for DSM-5, and collect blood samples at each of these visits for agnostic profiling of metabolic biomarkers through an untargeted metabolomic screen, using the rapid hydrophilic interaction liquid chromatography ion mobility mass spectrometry method (RHIMMS). Participants will also complete remote assessments at 3 and 9 months after the baseline visit: Ecological Momentary Assessments to measure mental health, wrist actigraphy to measure rhythms of rest and activity, and continuous glucose monitoring to measure metabolic changes. Throughout the 12-month enrolment period, we will also measure objective markers of sleep using a radar sleep monitor (Somnofy). Using advanced statistical techniques and machine learning analysis, we will seek to better understand the mechanisms linking metabolic health with mental health in young adults with schizophrenia, bipolar disorder, and severe depression. Clinical trial number: Not applicable

Glucocorticoids play a crucial role in the stress response and in the regulation of circadian function, as well as cognitive, cardiovascular, metabolic and immunological processes. While synthetic glucocorticoids (sGC) are widely used in treating inflammatory disorders, their impact on cognitive functions, which include memory deficits and dysregulation of mood, remains less understood. Here, we demonstrate that chronic treatment with the sGC methylprednisolone (MPL) dysregulates the synaptic proteome and impairs cognitive function in the perirhinal cortex, a region critical for recognition memory and visual perception. Further, we show that synaptic structure and plasticity are altered by MPL treatment, highlighting the mechanisms through which sGCs disrupt mnemonic processing. These results may have broad implications for understanding the cognitive side effects of widely used sGC treatments. ### Competing Interest Statement The authors have declared no competing interest.

Cortisol is released upon activation of the hypothalamic-pituitary-adrenal axis, varies across the day, possesses an underlying diurnal rhythm and is responsive to stressors. The endogenous circadian peak of cortisol occurs in the morning, and increases in cortisol observed post-awakening have been named the cortisol awakening response (CAR) based on the belief that the act of waking up stimulates cortisol secretion. However, objective evidence that awakening induces cortisol secretion is limited. We used a mixed effects model with a linear spline fitted to the data to examine tissue-free cortisol measurements obtained from 201 healthy volunteers by automated ambulatory microdialysis before and after awakening in their home environments. We also examined rate of change of cortisol depending on sleep duration and relative timing. We found no evidence for a change in the rate of cortisol increase in the hour after waking when compared with the hour prior to waking. We instead observed substantial interindividual variability in the absolute concentration and rate of change of cortisol levels, and differences in dynamics that may be attributable to duration and relative timing of sleep. Based on these results, we strongly suggest caution is needed when interpreting cortisol measurements solely obtained in the hour after waking.

We present a molecular mechanism underpinning how pulsatile patterns of glucocorticoid hormones maintain signal responsivity, evade hormone resistance, and promote homeostasis. Endogenous glucocorticoids are released in a pulsatile manner resulting in oscillating hormone signals with intermittent peaks of high glucocorticoids and troughs of low glucocorticoids. We show that ligand activation of glucocorticoid receptors rapidly triggers the post-translational modification SUMOylation, which is coupled to receptor degradation, whereby resistance to subsequent signal transduction is generated and ligand response attenuated. We find rapid, transient glucocorticoid receptor SUMOylation tracks ultradian (roughly hourly) pulse dynamics in cells, as well as circadian (daily) oscillatory rhythms in vivo, enabling cellular interpretation of fluctuating hormone patterns. Prolonged treatment with the long-acting synthetic glucocorticoid methylprednisolone disrupted glucocorticoid receptor SUMOylation levels in rat brain tissue. Pharmacological glucocorticoid therapy generates unremitting glucocorticoid signaling, which may substantially reduce the glucocorticoid receptor pool and contribute to the therapeutic problem of acquired glucocorticoid resistance. The physiological solution for maintaining signal responsivity over time is pulsatile hormone exposure, with pulsatile low glucocorticoid troughs which periodically limit receptor degradation and associated signal attenuation. We show low glucocorticoid periods allow time for depleted glucocorticoid receptor expression levels to recover and thereby maintain signal sensitivity. Our results reveal a molecular mechanism responsive to hormone pattern information, through which endogenous ultradian and circadian glucocorticoid fluctuations maintain glucocorticoid receptor expression and glucocorticoid sensitivity. Dynamic ligand-activated glucocorticoid receptor SUMOylation coupled to degradation is revealed as a component of glucocorticoid receptor protein regulation, whose expression is critical for metabolic, immunological, cognitive, and cardiovascular homeostasis.

Mineralocorticoid (MR) and glucocorticoid receptors (GR) act as transcription factors and major mediators of glucocorticoid signalling, with pivotal roles in regulating the stress response and hormonal signalling, mood, cognition and memory. The MR and GR share many target genes, have a high degree of homology in their DNA binding (DBD) and ligand binding domain (LBD) but differ considerably in the N-terminal domain (NTD). Using Proximity Ligation Assay (PLA) we quantitatively assessed MR-GR complex subcellular localisation and transcriptional regulation in murine neuroblastoma (N2A) cells stimulated by constant or pulsatile corticosterone (CORT) patterns. We observe that continuous receptor activation by CORT caused localisation at the periphery of the cell nucleus. Truncation of the receptor Ligand Binding Domain (LBD) led to a stronger localisation of MR-GR complexes at the periphery of the cell nuclei. This was also observed for GR immunofluorescence (IF), while in cells expressing only MR or GR the mRNA response to pulsatile hormone treatment was substantially attenuated. However, there was no clearcut correlation between the spatial distribution of MR-GR complexes and the mRNA levels of target genes. Overall, our findings suggest that longer presence in the cell nucleus favors more peripheral nuclear localisation.

The endocrine system consists of specialized secretory organs that exert their effects by direct release of hormones into the circulation. Hormones are secreted in pulsatile, rhythmic patterns, determined by endogenous pacemakers and complex feedback systems. The effect on target organs is to regulate growth, metabolism, and the response to stress. Endocrine rhythms create a coordinated, dynamic system of interacting processes that when disrupted result in disease.

Background. Primary adrenal insufficiency (PAI) mortality and morbidity remain unacceptably high, possibly arising as glucocorticoid replacement does not replicate natural physiology. A pulsatile subcutaneous pump can closely replicate cortisol's circadian and ultradian rhythm.Objectives. To assess the effect of pump therapy on quality of life, mood, functional neuroimaging, behavioural/cognitive responses, sleep and metabolism.Methods. A 6-week randomised, crossover, double-blinded and placebo-controlled feasibility study of usual dose hydrocortisone in PAI administered as either pulsed subcutaneous or standard care in Bristol, United Kingdom (ISRCTN67193733). Participants were stratified by adrenal insufficiency type. All participants who received study drugs are included in the analysis. The primary outcome, the facial expression recognition task (FERT), occurred at week 6.Results. Between December 2014 and 2017, 22 participants were recruited - 20 completed both arms, and 21 were analysed. The pump was well-tolerated. No change was seen in the FERT primary outcome; however, there were subjective improvements in fatigue and mood. Additionally, functional magnetic resonance imaging revealed differential neural processing to emotional cues and visual stimulation. Region of interest analysis identified the left amygdala and insula, key glucocorticoid-sensitive regions involved in emotional ambiguity. FERT post hoc analysis confirmed this response. There were four serious adverse events (AE): three intercurrent illnesses requiring hospitalisation (1/3, 33.3% pump) and a planned procedure (1/1, 100% pump). There was a small number of expected AEs: infusion site bruising/itching (3/5, 60% pump), intercurrent illness requiring extra (3/7, 42% pump) and no extra (4/6, 66% pump) steroid.Conclusions. These findings support the administration of hormone therapy that mimics physiology.

Diurnal variations in indicators of emotion have been reliably observed in Twitter content, but confirmation of their circadian nature has not been possible due to the many confounding factors present in the data. We report on correlations between those indicators in Twitter content obtained from 9 cities of Italy and 54 cities in the United Kingdom, sampled hourly at the time of the 2020 national lockdowns. This experimental setting aims at minimizing synchronization effects related to television, eating habits, or other cultural factors. This correlation supports a circadian origin for these diurnal variations, although it does not exclude the possibility that similar zeitgebers exist in both countries including during lockdowns.

The hypothalamic-pituitary-adrenal axis is an extremely dynamic system with a combination of both circadian and ultradian oscillations. This state of 'continuous dynamic equilibration' provides a platform that is able to anticipate events, is sensitive in its response to stressors, remains robust during perturbations of both the internal and external environments and shows plasticity to adapt to a changed environment. In this review, we describe these oscillations of glucocorticoid (GC) hormones and why they are so important for GC-dependent gene activation in the brain and liver, and their consequent effects on the regulation of synaptic and memory function as well as appetite control and metabolic regulation. Abnormalities of mood, appetite and metabolic regulation are well-known consequences of GC therapy, and we suggest that the pattern of GC treatment and hormone replacement should be a much higher priority for endocrinologists and the pharmaceutical industry. One of the major impediments to our research on the importance of these cortisol rhythms in our patients has been our inability to measure repeated levels of hormones across the day in patients in their home or work surroundings. We describe how new wearable methodologies now allow the measurement of 24-h cortisol profiles - including during sleep - and will enable us to define physiological normality and allow us both to develop better diagnostic tests and inform, at an individual patient level, how to improve replacement therapy.

Introduction: Traumatic brain injury (TBI) is a leading cause of acquired neurological morbidity. The prevalence of post-traumatic hypopituitarism and associated morbidity after childhood TBI is unclear. Our study investigated long-term hypothalamus-pituitary-adrenal (HPA) axis function, in a prospective childhood TBI and control cohort, using measures of cortisol/cortisone secretion (physiological and stimulated), HPA axis feedback, and exploring associations with fatigue, depression, and quality of life (QoL) outcomes. Methods: All TBI participants had data concerning severity and mechanism of TBI. All groups had clinical assessment, pituitary/brain MRI, questionnaire measures of QoL, fatigue, depression, and salivary cortisone profiles including dexamethasone suppression test. In addition, participants with moderate/severe TBI had ethical approval for baseline endocrine blood tests, overnight 12-h venous sampling of cortisol and growth hormone, and stimulated HPA axis evaluation with an insulin tolerance test (ITT). Results: Seventy-two participants with moderate/severe (n = 31, age 19.8 ± 4.2 years) or mild TBI (n = 24, age 17.8 ± 5.1 years) and matched controls (n = 17, age 18.5 ± 5.5 years) took part. Time post-TBI was 6.8–10.8 years. Baseline endocrine tests confirmed normal thyroid and posterior pituitary function. One female with moderate/severe TBI had hypogonadism. Pituitary neuroimaging was normal in all participants. In 2/25 ITT and 9/22 overnight serum profiles, peak cortisol was <500 nmol/L. The two participants with suboptimal ITT cortisol response (392 and 483 nmol/L) also had low peak spontaneous serum levels (227 and 447 nmol/L, respectively). Salivary cortisone profiles showed preservation of HPA axis circadian rhythm and suppression with dexamethasone in all but one TBI participant. TBI participants had higher morning salivary cortisone levels compared to controls. Fatigue was reported by 20/46 TBI participants but only 1/14 controls. Fatigue was not associated with stimulated (ITT) or spontaneous (overnight profile) cortisol; however, one TBI participant with severe fatigue had a suboptimal ITT cortisol response. Specific QoL attributes of health state (cognition, memory) were impaired in TBI participants compared to controls. Conclusion: Although not as prevalent as previously reported, HPA axis dysfunction does occur in survivors of childhood TBI, confirming the need for endocrine surveillance. However, in most of our paediatric TBI survivors assessed 7–11 years post-TBI, HPA function and circadian rhythmicity were preserved or had recovered. Chronic fatigue is a common concern post-TBI, but in the majority, it is not associated with frank HPA axis dysfunction. Morning salivary cortisone levels were higher in TBI survivors (who have a high prevalence of fatigue) compared to healthy controls, despite the recognised association of chronic fatigue with cortisol hyposecretion.

Selye described stress as a unified neurohormonal mechanism maintaining homeostasis. Acute stress system activation is adaptive through neurocognitive, catecholaminergic, and immunomodulation mechanisms, followed by a reset via cortisol. Stress system components, the sympathoadrenomedullary system, hypothalamic-pituitary-adrenal axis, and limbic structures are implicated in many chronic diseases by establishing an altered homeostatic state, allostasis. Consequent "primary stress system disorders" were popularly accepted, with phenotypes based on conditions such as Cushing syndrome, pheochromocytoma, and adrenal insufficiency. Cardiometabolic and major depressive disorders are candidates for hypercortisolemic etiology, contrasting the "hypocortisolemic symptom triad" of stress sensitivity, chronic fatigue, and pain. However, acceptance of chronic stress etiology requires cause-and-effect associations, and practical utility such as therapeutics altering stress system function. Inherent predispositions to stress system perturbations may be relevant. Glucocorticoid receptor (GR) variants have been associated with metabolic/neuropsychological states. The SERPINA6 gene encoding corticosteroid-binding globulin (CBG), was the sole genetic factor in a single-nucleotide variation-genome-wide association study linkage study of morning plasma cortisol, a risk factor for cardiovascular disease, with alterations in tissue-specific GR-related gene expression. Studies showed genetically predicted high cortisol concentrations are associated with hypertension and anxiety, and low CBG concentrations/binding affinity, with the hypocortisolemic triad. Acquired CBG deficiency in septic shock results in 3-fold higher mortality when hydrocortisone administration produces equivocal results, consistent with CBG's role in spatiotemporal cortisol delivery. We propose some stress system disorders result from constitutional stress system variants rather than stressors themselves. Altered CBG:cortisol buffering may influence interstitial cortisol ultradian surges leading to pathological tissue effects, an example of stress system variants contributing to stress-related disorders.

Measurement of blood levels of circulating hormones has always been the cornerstone of the biochemical diagnosis of endocrine diseases, with the objective of detecting hormone excess or insufficiency. Unfortunately, the dynamic nature of hormone secretion means single-point measurements of many hormones often lack diagnostic validity. Endocrinologists have devised complex dynamic tests as indirect assessments of the functioning of the hormone system under investigation. Recent advances in the measurement of dynamic hormone changes across the day now offer an opportunity to reconsider whether there might be better ways both to diagnose and to monitor the therapy of endocrine conditions.

Rhythmicity is a intrinsic feature of biological systems, including the hypothalamic-pituitary-adrenal axis, a mammalian neurohormonal system crucial both in daily life and as a network that responds to stressful stimuli. Circadian and ultradian rhythmicity underlie HPA activity in rodents and in humans, regulating gene expression, metabolism and behavior, and adverse consequences occur when rhythms are disturbed. In the assessment of human disease, the complexity of HPA rhythmicity is rarely acknowledged or understood, and is currently a limitation to better diagnosis and treatment. However, the recent emergence of ambulatory, high frequency and blood-free hormone sampling techniques has the promise to substantially change our understanding of the function of HPA axis in healthy normal life, and provide new opportunities for the diagnosis and treatment of disease.

The hormone cortisol, released as the end-product of the hypothalamic-pituitary-adrenal (HPA) axis, has a well-characterized circadian rhythm that enables an allostatic response to external stressors. When the pattern of secretion is disrupted, cortisol levels are chronically elevated, contributing to diseases such as heart attacks, strokes, mental health disorders, and diabetes. The diagnosis of chronic stress and stress related disorders depends upon accurate measurement of cortisol levels; currently, it is quantified using mass spectroscopy or immunoassay, in specialized laboratories with trained personnel. However, these methods are time-consuming, expensive and are unable to capture the dynamic biorhythm of the hormone. This critical review traces the path of cortisol detection from traditional laboratory-based methods to decentralised cortisol monitoring biosensors. A complete picture of cortisol biology and pathophysiology is provided, and the importance of precision medicine style monitoring of cortisol is highlighted. Antibody-based immunoassays still dominate the pipeline of development of point-of-care biosensors; new capture molecules such as aptamers and molecularly imprinted polymers (MIPs) combined with technologies such as microfluidics, wearable electronics, and quantum dots offer improvements to limit of detection (LoD), specificity, and a shift toward rapid or continuous measurements. While a variety of different sensors and devices have been proposed, there still exists a need to produce quantitative tests for cortisol ─ using either rapid or continuous monitoring devices that can enable a personalized medicine approach to stress management. This can be addressed by synergistic combinations of technologies that can leverage low sample volumes, relevant limit of detection and rapid testing time, to better account for cortisol's shifting biorhythm. Trends in cortisol diagnostics toward rapid and continuous monitoring of hormones are highlighted, along with insights into choice of sample matrix.

Cortisol is the primary glucocorticoid hormone produced by the adrenal glands, playing a critical role in multiple physiological processes, including metabolism, immune response regulation, cardiovascular regulation, and the body's stress response1Russell G Lightman S. The human stress response.Nat Rev Endocrinol. 2019; 15: 525-534Crossref PubMed Scopus (388) Google Scholar. The secretion of cortisol is closely regulated by the hypothalamus-pituitary-adrenal (HPA) axis. The process is initiated by the release of corticotropin-releasing hormone (CRH) from the hypothalamus, which stimulates the production of adrenocorticotropic hormone (ACTH) by the pituitary gland. In turn, ACTH prompts the adrenal glands to release cortisol1Russell G Lightman S. The human stress response.Nat Rev Endocrinol. 2019; 15: 525-534Crossref PubMed Scopus (388) Google Scholar. Cortisol measurements have been used extensively in research to investigate the hormone's relationship with various physiological disruptions and health conditions, including mental health disorders, immune system dysfunction, and metabolic disorders2Lovallo WR Buchanan TW. Stress hormones in psychophysiological research: Emotional, behavioral, and cognitive implications.in: Cacioppo JT Tassinary LG Berntson GG Handbook of psychophysiology. Cambridge University Press, 2017: 465-494Google Scholar. However, it is essential to recognize the potential pitfalls in the assessment and interpretation of cortisol levels in translational research, as overlooking them can foster misinformation and confusion regarding potential clinical or diagnostic relevance of the findings. In two recent studies (Klein et al. in 20233Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar and Fleischer et al. in 20244Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar), researchers reported on the analysis of hormonal and other markers in blood samples from individuals with post-acute sequelae of SARS CoV-2 infection (PASC)—commonly known as long COVID (LC). Long COVID is associated with a range of persistent symptoms, including fatigue, pain, and brain fog that can be debilitating. Notably, the most compelling and statistically significant finding of the Klein et al. study revolved around the levels of cortisol, which were reported to be much lower in long COVID patients than in controls without long COVID3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar. The authors of the study concluded that "[s]erum cortisol was the most significant predictor of LC status in the model, and cortisol alone achieved an AUC [area under curve] of 0.96"3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar. The findings have been widely publicized in the media, engendering speculation regarding their therapeutic and biomarker potential in long COVID5Goodman B. Scientists edge closer to finding a biomarker for long Covid, which could lead to better tests and treatments.: CNN Health, 25 September 2023. Available at: https://www.cnn.com/2023/09/25/health/long-covid-studies-test-treatment-development-wellness/index.html. Accessed 20 December 2023.Google Scholar,6Edwards E. A blood test for long Covid is possible, a study suggests. NBC News, 2023https://www.nbcnews.com/health/health-news/long-covid-differences-blood-test-study-finds-rcna116871Google Scholar. On the other hand, a more recent study by Fleischer et al.4Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar found no significant difference or trends in the cortisol levels in a comparison of post-COVID-19 patients, with and without persistent symptoms, apparently contradicting the results reported in the earlier study3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar. While the cortisol assessments in both of these two studies3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar,4Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar may represent remarkable and consequential findings in the context of long COVID, unfortunately, they are impacted by significant limitations and concerns regarding how the cortisol levels were assessed and interpreted. The levels of cortisol in the body are very dynamic, following a distinct circadian pattern throughout the day. Levels typically increase in the first half hour after awakening by up to 60%, in what has been called the cortisol awakening response, followed by a sharp drop over the next few hours, reaching their lowest point in the evening7Adam EK Hawkley LC Kudielka BM et al.Day-to-day dynamics of experience–cortisol associations in a population-based sample of older adults.Proc Natl Acad Sci U S A. 2006; 103: 17058-17063Crossref PubMed Scopus (594) Google Scholar. Underlying this circadian rhythm, cortisol also exhibits ultradian fluctuations in a pulsatile pattern with several peaks and troughs throughout the day that change in amplitude1Russell G Lightman S. The human stress response.Nat Rev Endocrinol. 2019; 15: 525-534Crossref PubMed Scopus (388) Google Scholar. Because of these fluctuations and the wide and rapid changes in circulating levels, reliable and reproducible evaluation of cortisol secretion is not trivial. Such assessment cannot be based on only a single measurement, but rather requires the collection of multiple blood, saliva, or urine samples throughout the day. Any single measurement would also ideally need to account for the time elapsed since awakening and not necessarily just the time of day. In assessing the difference in cortisol output between groups, multiple measurements in blood or saliva samples are usually used to analyze the change from baseline, the size of the cortisol awakening response, or the elevation and slope of the diurnal curve2Lovallo WR Buchanan TW. Stress hormones in psychophysiological research: Emotional, behavioral, and cognitive implications.in: Cacioppo JT Tassinary LG Berntson GG Handbook of psychophysiology. Cambridge University Press, 2017: 465-494Google Scholar. Surprisingly, none of these approaches was taken in either of the two cited studies of cortisol levels in long COVID3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar,4Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar. Both studies conducted a single measurement in blood plasma or serum, taking note of the time of day, but not the time since awakening, for the sample collection. More importantly, no attempt was made to take serial samples to overcome the problem associated with the very rapid changes occurring due to ultradian rhythmicity. Considering the dramatic ultradian and diurnal cortisol oscillations, meaningful interpretation of the reported cortisol levels in these two studies3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar,4Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar is simply not possible. The issue is further compounded by the potential association of long COVID with sleep disturbances8Pena-Orbea C Lapin B Li Y et al.Sleep disturbance severity and correlates in post-acute sequelae of COVID-19 (PASC).J Gen Intern Med. 2023; 38: 2015-2017Crossref Scopus (2) Google Scholar, including earlier than average awakening times, which could significantly shift the cortisol diurnal curve, giving the appearance of uniformly lower circulating levels during the daytime. The additional finding that the time of sample collection was not a significant predictor of cortisol levels in the study by Klein et al.3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar adds weight to existing concerns about the validity of the reported results and the conclusions regarding the value of blood cortisol concentration as a potential biomarker of long COVID. Given the presence of persistent fatigue as a hallmark symptom of long COVID, the evaluation of HPA axis function through the assessment of cortisol output is a logical approach. However, considering the available data and the major methodological shortcomings noted in the two published studies3Klein J Wood J Jaycox J et al.Distinguishing features of Long COVID identified through immune profiling.Nature. 2023; 623: 139-148Crossref PubMed Scopus (20) Google Scholar,4Fleischer M Szepanowski F Mausberg AK et al.Cytokines (IL1beta, IL6, TNFalpha) and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19.Ther Adv Neurol Disord. 2024; 1717562864241229567Crossref Scopus (0) Google Scholar, it is clear that neither the utility of cortisol as a biomarker nor the therapeutic role of corticosteroids in treating long COVID can be justified at this juncture. The issues raised here underscore the need for further rigorous and comprehensive investigations into the relationship between cortisol response and long COVID, taking special note of the cortisol diurnal pattern, potential confounding factors impacting this pattern, and the utilization of well-documented and established methods of assessing cortisol levels9El-Farhan N Rees DA Evans C. Measuring cortisol in serum, urine and saliva - are our assays good enough?.Ann Clin Biochem. 2017; 54: 308-322Crossref PubMed Scopus (0) Google Scholar. The recent availability of new techniques to measure cortisol levels sequentially across the full 24 hours, including during sleep10Upton TJ Zavala E Methlie P et al.High-resolution daily profiles of tissue adrenal steroids by portable automated collection.Sci Transl Med. 2023; 15: eadg8464Crossref Scopus (8) Google Scholar, should provide the methodology needed for obtaining reliable and accurate data on HPA axis function in long COVID and other conditions. All authors contributed to the writing and approval of the manuscript. AA reports participation on advisory panels for the National Institutes of Health, Global Lyme Alliance, Roche, Everlywell, and Veravas. SL is a co-founder of Dynamic Therapeutics. GPW reports research grants from Pfizer, Inc, and Biopeptides, Corp; has been an expert witness in malpractice cases involving Lyme disease and babesiosis; and is an unpaid board member of the non-profit American Lyme Disease Foundation. Armin Alaedini: Conceptualization, Writing – original draft, Writing – review & editing. Stafford Lightman: Conceptualization, Writing – review & editing. Gary P. Wormser: Conceptualization, Writing – review & editing. None None