Twelve-hour rhythms in transcript expression within the human dorsolateral prefrontal cortex are altered in schizophrenia.

Twelve-hour (12 h) ultradian rhythms are a well-known phenomenon in coastal marine organisms. While 12 h cycles are observed in human behavior and physiology, no study has measured 12 h rhythms in the human brain. Here, we identify 12 h rhythms in transcripts that either peak at sleep/wake transitions (approximately 9 AM/PM) or static times (approximately 3 PM/AM) in the dorsolateral prefrontal cortex, a region involved in cognition. Subjects with schizophrenia (SZ) lose 12 h rhythms in genes associated with the unfolded protein response and neuronal structural maintenance. Moreover, genes involved in mitochondrial function and protein translation, which normally peak at sleep/wake transitions, peak instead at static times in SZ, suggesting suboptimal timing of these essential processes.

A mathematical model for the role of dopamine-D2 self-regulation in the production of ultradian rhythms.

Many self-motivated and goal-directed behaviours display highly flexible, approximately 4 hour ultradian (shorter than a day) oscillations. Despite lacking direct correspondence to physical cycles in the environment, these ultradian rhythms may be involved in optimizing functional interactions with the environment and reflect intrinsic neural dynamics. Current evidence supports a role of mesostriatal dopamine (DA) in the expression and propagation of ultradian rhythmicity, however, the biochemical processes underpinning these oscillations remain to be identified. Here, we use a mathematical model to investigate D2 autoreceptor-dependent DA self-regulation as the source of ultradian behavioural rhythms. DA concentration at the midbrain-striatal synapses is governed through a dual-negative feedback-loop structure, which naturally gives rise to rhythmicity. This model shows the propensity of striatal DA to produce an ultradian oscillation characterized by a flexible period that is highly sensitive to parameter variations. Circadian (approximately 24 hour) regulation consolidates the ultradian oscillations and alters their response to the phase-dependent, rapid-resetting effect of a transient excitatory stimulus. Within a circadian framework, the ultradian rhythm orchestrates behavioural activity and enhances responsiveness to an external stimulus. This suggests a role for the circadian-ultradian timekeeping hierarchy in governing organized behaviour and shaping daily experience through coordinating the motivation to engage in recurring, albeit not highly predictable events, such as social interactions.

Coupling-dependent metabolic ultradian rhythms in confluent cells.

Ultradian rhythms in metabolism and physiology have been described previously in mammals. However, the underlying mechanisms for these rhythms are still elusive. Here, we report the discovery of temperature-sensitive ultradian rhythms in mammalian fibroblasts that are independent of both the cell cycle and the circadian clock. The period in each culture is stable over time but varies in different cultures (ranging from 3 to 24 h). We show that transient, single-cell metabolic pulses are synchronized into stable ultradian rhythms across contacting cells in culture by gap junction-mediated coupling. Coordinated rhythms are also apparent for other metabolic and physiological measures, including plasma membrane potential (Δψp), intracellular glutamine, α-ketoglutarate, intracellular adenosine triphosphate (ATP), cytosolic pH, and intracellular calcium. Moreover, these ultradian rhythms require extracellular glutamine, several different ion channels, and the suppression of mitochondrial ATP synthase by α-ketoglutarate, which provides a key feedback mechanism. We hypothesize that cellular coupling and metabolic feedback can be used by cells to balance energy demands for survival.

Circadian and ultradian rhythms: Clinical implications.

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.

Ultradian hydrocortisone replacement alters neuronal processing, emotional ambiguity, affect and fatigue in adrenal insufficiency: The PULSES trial.

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.

Beyond circadian rhythms: emerging roles of ultradian rhythms in control of liver functions.

The mammalian liver must cope with various metabolic and physiological changes that normally recur every day and primarily stem from daily cycles of rest-activity and fasting-feeding. Although a large body of evidence supports the reciprocal regulation of circadian rhythms and liver function, the research on the hepatic ultradian rhythms have largely been lagging behind. However, with the advent of more cost-effective high-throughput omics technologies, high-resolution time-lapse imaging, and more robust and powerful mathematical tools, several recent studies have shed new light on the presence and functions of hepatic ultradian rhythms. In this review, we will first very briefly discuss the basic principles of circadian rhythms, and then cover in greater details the recent literature related to ultradian rhythms. Specifically, we will highlight the prevalence and mechanisms of hepatic 12-h rhythms, and 8-h rhythms, which cycle at the second and third harmonics of circadian frequency. Finally, we also refer to ultradian rhythms with other frequencies and examine the limitations of the current approaches as well as the challenges related to identifying ultradian rhythm and addressing their molecular underpinnings.

Co-expression of diurnal and ultradian rhythms in the plasma metabolome of common voles (Microtus arvalis).

Metabolic rhythms include rapid, ultradian (hourly) dynamics, but it remains unclear what their relationship to circadian metabolic rhythms is, and what role meal timing plays in coordinating these ultradian rhythms in metabolism. Here, we characterized widespread ultradian rhythms under ad libitum feeding conditions in the plasma metabolome of the vole, the gold standard animal model for behavioral ultradian rhythms, naturally expressing ~2-h foraging rhythms throughout the day and night. These ultradian metabolite rhythms co-expressed with diurnal 24-h rhythms in the same metabolites and did not align with food intake patterns. Specifically, under light-dark entrained conditions we showed twice daily entrainment of phase and period of ultradian behavioral rhythms associated with phase adjustment of the ultradian cycle around the light-dark and dark-light transitions. These ultradian activity patterns also drove an ultradian feeding pattern. We used a unique approach to map this behavioral activity/feeding status to high temporal resolution (every 90 min) measures of plasma metabolite profiles across the 24-h light-dark cycle. A total of 148 known metabolites were detected in vole plasma. Supervised, discriminant analysis did not group metabolite concentration by feeding status, instead, unsupervised clustering of metabolite time courses revealed clusters of metabolites that exhibited significant ultradian rhythms with periods different from the feeding cycle. Two clusters with dissimilar ultradian dynamics, one lipid-enriched (period = 3.4 h) and one amino acid-enriched (period = 4.1 h), both showed co-expression with diurnal cycles. A third cluster solely comprised of glycerophospholipids (specifically ether-linked phosphatidylcholines) expressed an 11.9 h ultradian rhythm without co-expressed diurnal rhythmicity. Our findings show coordinated co-expression of diurnal metabolic rhythms with rapid dynamics in feeding and metabolism. These findings reveal that ultradian rhythms are integral to biological timing of metabolic regulation, and will be important in interpreting the impact of circadian desynchrony and meal timing on metabolic rhythms.

Ultradian rhythms of AKT phosphorylation and gene expression emerge in the absence of the circadian clock components Per1 and Per2.

Rhythmicity of biological processes can be elicited either in response to environmental cycles or driven by endogenous oscillators. In mammals, the circadian clock drives about 24-hour rhythms of multitude metabolic and physiological processes in anticipation to environmental daily oscillations. Also at the intersection of environment and metabolism is the protein kinase-AKT. It conveys extracellular signals, primarily feeding-related signals, to regulate various key cellular functions. Previous studies in mice identified rhythmicity in AKT activation (pAKT) with elevated levels in the fed state. However, it is still unknown whether rhythmic AKT activation can be driven through intrinsic mechanisms. Here, we inspected temporal changes in pAKT levels both in cultured cells and animal models. In cultured cells, pAKT levels showed circadian oscillations similar to those observed in livers of wild-type mice under free-running conditions. Unexpectedly, in livers of Per1,2-/- but not of Bmal1-/- mice we detected ultradian (about 16 hours) oscillations of pAKT levels. Importantly, the liver transcriptome of Per1,2-/- mice also showed ultradian rhythms, corresponding to pAKT rhythmicity and consisting of AKT-related genes and regulators. Overall, our findings reveal ultradian rhythms in liver gene expression and AKT phosphorylation that emerge in the absence of environmental rhythms and Per1,2-/- genes.

A Method for Predicting Ultradian Body Temperature Rhythms in Small Animals.

It has been found that the intraday dynamics of body temperature in small mammal and bird species on the adjacent day are similar. Therefore, by focusing on the body temperature dynamics of the previous day, it is possible to predict with a high degree of accuracy the periods of increase and decrease in body temperature for the current day. This phenomenon was observed when animals were kept under natural illumination and under artificial illumination when the phase of the intrinsic circadian rhythm shifted by 1-2 h every day. When analyzing this phenomenon in birds, it has been shown that the best match for body temperature dynamics occurs when comparing adjacent days based on sidereal days (a period of 23 h and 56 min). Over time, after several days, the daily patterns of body temperature fluctuation take on a completely different form and frequency. These facts suggest a connection between ultradian rhythms and the rotation of the Earth around its axis, and consequently, the position of animals on the surface of the planet relative to space objects.

Association between Ultradian Rhythms of Body Temperature in Small Mammals and Earth's Crust Stress.

Association was assessed between the data harvested by a long-baseline laser interference deformograph and the dynamics of body temperature (BT) in hamsters deprived of natural daily light-darkness changes. The power spectral data revealed the positive correlation between simultaneous time series of hamster BT and the Earth's crust deformation (ECD). The superposed epoch analysis established an association between abrupt upstrokes of hamster BT and ECD increments. Thus, the direct relationships between BT dynamics (reflecting predominance of sympathetic part of autonomic nervous system) and ECD (according to long-baseline laser interference deformography) were established. The study observed synchronization of the free-running circadian rhythm of hamster BT with the tidal stress in Earth's lithosphere. Further studies are needed to find the physical factor underlying the revealed relationships.

DEC-7/SUSD2, a sushi domain-containing protein, regulates an ultradian behavior mediated by intestinal epithelial Ca2+ oscillations in Caenorhabditis elegans.

In Caenorhabditis elegans, rhythmic posterior body wall muscle contractions mediate the highly regular defecation cycle. These contractions are regulated by inositol-1,4,5-trisphosphate (InsP3) receptor-dependent Ca2+ oscillations in intestinal epithelial cells. Here, we find that mutations in dec-7, which encodes the nematode ortholog of the human Sushi domain-containing 2 protein (SUSD2), lead to an increase in InsP3 receptor-dependent rhythmic posterior body wall muscle contractions. DEC-7 is highly expressed in the intestinal epithelia and localizes to the cell-cell junction. The increase in rhythmic activity caused by the loss of dec-7 is dependent on the innexin gap junction protein INX-16. Moreover, DEC-7 is required for the clustering of INX-16 to the cell-cell junction of the intestinal epithelia. We hypothesize that DEC-7/SUSD2 regulates INX-16 activity to mediate the rhythmic frequency of the defecation motor program. Thus, our data indicate a critical role of a phylogenetically conserved cell-cell junction protein in mediating an ultradian rhythm in the intestinal epithelia of C. elegans.NEW & NOTEWORTHY The conserved complement group protein DEC-7/SUSD2 acts at the apical cell-cell junction of C. elegans intestinal epithelia to mediate gap junction protein organization and function to facilitate a Ca2+ wave-regulated ultradian behavior.

Peroxiredoxins couple metabolism and cell division in an ultradian cycle.

Redox cycles have been reported in ultradian, circadian and cell cycle-synchronized systems. Redox cycles persist in the absence of transcription and cyclin-CDK activity, indicating that cells harbor multiple coupled oscillators. Nonetheless, the causal relationships and molecular mechanisms by which redox cycles are embedded within ultradian, circadian or cell division cycles remain largely elusive. Yeast harbor an ultradian oscillator, the yeast metabolic cycle (YMC), which comprises metabolic/redox cycles, transcriptional cycles and synchronized cell division. Here, we reveal the existence of robust cycling of H2O2 and peroxiredoxin oxidation during the YMC and show that peroxiredoxin inactivation disrupts metabolic cycling and abolishes coupling with cell division. We find that thiol-disulfide oxidants and reductants predictably modulate the switching between different YMC metabolic states, which in turn predictably perturbs cell cycle entry and exit. We propose that oscillatory H2O2-dependent protein thiol oxidation is a key regulator of metabolic cycling and its coordination with cell division.

Budgerigars have complex sleep structure similar to that of mammals.

Birds and mammals share specialized forms of sleep including slow wave sleep (SWS) and rapid eye movement sleep (REM), raising the question of why and how specialized sleep evolved. Extensive prior studies concluded that avian sleep lacked many features characteristic of mammalian sleep, and therefore that specialized sleep must have evolved independently in birds and mammals. This has been challenged by evidence of more complex sleep in multiple songbird species. To extend this analysis beyond songbirds, we examined a species of parrot, the sister taxon to songbirds. We implanted adult budgerigars (Melopsittacus undulatus) with electroencephalogram (EEG) and electrooculogram (EOG) electrodes to evaluate sleep architecture, and video monitored birds during sleep. Sleep was scored with manual and automated techniques, including automated detection of slow waves and eye movements. This can help define a new standard for how to score sleep in birds. Budgerigars exhibited consolidated sleep, a pattern also observed in songbirds, and many mammalian species, including humans. We found that REM constituted 26.5% of total sleep, comparable to humans and an order of magnitude greater than previously reported. Although we observed no spindles, we found a clear state of intermediate sleep (IS) similar to non-REM (NREM) stage 2. Across the night, SWS decreased and REM increased, as observed in mammals and songbirds. Slow wave activity (SWA) fluctuated with a 29-min ultradian rhythm, indicating a tendency to move systematically through sleep states as observed in other species with consolidated sleep. These results are at variance with numerous older sleep studies, including for budgerigars. Here, we demonstrated that lighting conditions used in the prior budgerigar study-and commonly used in older bird studies-dramatically disrupted budgerigar sleep structure, explaining the prior results. Thus, it is likely that more complex sleep has been overlooked in a broad range of bird species. The similarities in sleep architecture observed in mammals, songbirds, and now budgerigars, alongside recent work in reptiles and basal birds, provide support for the hypothesis that a common amniote ancestor possessed the precursors that gave rise to REM and SWS at one or more loci in the parallel evolution of sleep in higher vertebrates. We discuss this hypothesis in terms of the common plan of forebrain organization shared by reptiles, birds, and mammals.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Physiological Rhythms and Biological Variation of Biomolecules: The Road to Personalized Laboratory Medicine.

The concentration of biomolecules in living systems shows numerous systematic and random variations. Systematic variations can be classified based on the frequency of variations as ultradian (<24 h), circadian (approximately 24 h), and infradian (>24 h), which are partly predictable. Random biological variations are known as between-subject biological variations that are the variations among the set points of an analyte from different individuals and within-subject biological variation, which is the variation of the analyte around individuals' set points. The random biological variation cannot be predicted but can be estimated using appropriate measurement and statistical procedures. Physiological rhythms and random biological variation of the analytes could be considered the essential elements of predictive, preventive, and particularly personalized laboratory medicine. This systematic review aims to summarize research that have been done about the types of physiological rhythms, biological variations, and their effects on laboratory tests. We have searched the PubMed and Web of Science databases for biological variation and physiological rhythm articles in English without time restrictions with the terms "Biological variation, Within-subject biological variation, Between-subject biological variation, Physiological rhythms, Ultradian rhythms, Circadian rhythm, Infradian rhythms". It was concluded that, for effective management of predicting, preventing, and personalizing medicine, which is based on the safe and valid interpretation of patients' laboratory test results, both physiological rhythms and biological variation of the measurands should be considered simultaneously.

Analysis of Association of Ultradian Body Temperature Rhythms in Animals with Intensity of Fluctuations of Radioactive Decay of Natural 40K Isotope.

The study monitored the long-term body temperature (BT) oscillations of C57BL/6 mice and outbred starlings (Sturnus vulgaris) to compare them with fluctuation in decay rate of radioactive natural 40K isotope. The spectrum analysis revealed simultaneous changes of the predominant periods in BT spectra of the animals and those in fluctuation in 40K decay rate. A positive correlation was established between BT dynamics and fluctuation in decay rate. The superposed epoch analysis revealed predominant coincidence of the moments of BT and fluctuation in 40K decay rate. The novel data indicate association between BT ultradian rhythms with quasirhythmic variations of fluctuation in 40K decay rate.

Effect of Heliogeophysical and Atmospheric Factors on the Degree of Synchronization of Ultradian Rhythms of Body Temperature in Mice.

In a long-term (8 months) study, we examined the degree of synchronization of ultradian body temperature oscillations of two isolated groups of mice kept under constant dim illumination. In most cases, the periods of increased activity accompanied by rapid elevation of body temperature coincided in these groups of mice, but in some days, no significant synchronization between the examined parameters was observed. Analysis of the effects of environmental factors on the degree of synchronization of ultradian rhythms in mice revealed association of this parameter with the dynamics of atmospheric pressure (AtmP) and to a lesser extent with the vertical component of interplanetary magnetic field Bz. The loss in synchronicity of ultradian rhythms of mouse activity occurred after a rapid drop of AtmP or during pronounced negative Bz. Therefore, these factors can be viewed as desynchronizers of the biological ultradian rhythms.

A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord.

Ultradian oscillations of HES Transcription Factors (TFs) at the single-cell level enable cell state transitions. However, the tissue-level organisation of HES5 dynamics in neurogenesis is unknown. Here, we analyse the expression of HES5 ex vivo in the developing mouse ventral spinal cord and identify microclusters of 4-6 cells with positively correlated HES5 level and ultradian dynamics. These microclusters are spatially periodic along the dorsoventral axis and temporally dynamic, alternating between high and low expression with a supra-ultradian persistence time. We show that Notch signalling is required for temporal dynamics but not the spatial periodicity of HES5. Few Neurogenin 2 cells are observed per cluster, irrespective of high or low state, suggesting that the microcluster organisation of HES5 enables the stable selection of differentiating cells. Computational modelling predicts that different cell coupling strengths underlie the HES5 spatial patterns and rate of differentiation, which is consistent with comparison between the motoneuron and interneuron progenitor domains. Our work shows a previously unrecognised spatiotemporal organisation of neurogenesis, emergent at the tissue level from the synthesis of single-cell dynamics.

Revealing the hidden reality of the mammalian 12-h ultradian rhythms.

Biological oscillations often cycle at different harmonics of the 24-h circadian rhythms, a phenomenon we coined "Musica Universalis" in 2017. Like the circadian rhythm, the 12-h oscillation is also evolutionarily conserved, robust, and has recently gained new traction in the field of chronobiology. Originally thought to be regulated by the circadian clock and/or environmental cues, recent new evidences support the notion that the majority of 12-h rhythms are regulated by a distinct and cell-autonomous pacemaker that includes the unfolded protein response (UPR) transcription factor spliced form of XBP1 (XBP1s). 12-h cycle of XBP1s level in turn transcriptionally generates robust 12-h rhythms of gene expression enriched in the central dogma information flow (CEDIF) pathway. Given the regulatory and functional separation of the 12-h and circadian clocks, in this review, we will focus our attention on the mammalian 12-h pacemaker, and discuss our current understanding of its prevalence, evolutionary origin, regulation, and functional roles in both physiological and pathological processes.

The relationship between early post-operative ACTH / cortisol following pituitary surgery and long-term glucocorticoid requirement - Do ultradian rhythms matter?

OBJECTIVE: To determine whether early (4-8h) post-operative ACTH after trans-sphenoidal surgery (TSS) predicts long-term hypothalamic-pituitary-adrenal (HPA) axis function and to investigate early morning day 1 ACTH/cortisol variability using rapid sampling. DESIGN: Prospective observational study. METHODS: Participants undergoing TSS were included; those treated with glucocorticoids pre-operatively received 100 mg intravenous hydrocortisone on anaesthetic induction. ACTH and cortisol were measured post-operatively at + 4h and + 8h after induction and on day 1 every 10 minutes between 0700h and 0900h. PRIMARY OUTCOME: glucocorticoid requirement at 6 months. RESULTS: Nineteen participants (10F, 9M): 6/19 (32%) were treated with replacement glucocorticoids pre-operatively; 4 had ceased by 6 weeks post-operatively. One patient developed new hypopituitarism post-operatively meaning 3/19 (16%) required glucocorticoids at 6 months. Post-operative + 4h ACTH < 14.3 pmol/L (65 ng/L) predicted secondary adrenal insufficiency (SAI) (sensitivity 100%, specificity 75%), whilst no participant with a post-operative + 4h ACTH ≥ 14.3 pmol/L (65 ng/L) required glucocorticoids at 6 months. Day 1 ACTH and cortisol showed a significant circadian fall between 0700h-0900h; ACTH 4.2 pmol/L (IQR 2.9-5.9) to 3.7 pmol/L (IQR 2.9-5.1) P = .006 and cortisol 549 nmol/L (IQR 337-618) to 439 nmol/L (IQR 315-606) P < .001, with clinically insignificant ultradian secretory pulses. CONCLUSIONS: No participant with a post-operative + 4h ACTH ≥ 14.3 pmol/L (65 ng/L) required glucocorticoids at 6 months; however, given only 3/19 participants had the primary outcome of interest, this must be confirmed in a larger cohort. The timing of a day 1 morning cortisol between 0700h and 0900h influences the accuracy of a single cut-off to diagnose SAI after pituitary surgery.