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This study aimed to analyze the associations of activity-rest pattern indicators with academic achievement, executive function, and intelligence and to explore whether these associations are mediated by the total gray matter volume among children with overweight/obesity. Ninety-five children (10 ± 1 year, 37 girls) with overweight/obesity (based on the World Obesity Federation body mass index cutoff points) were included in this cross-sectional study. Hip- and wrist-worn ActiGraph GT3X + accelerometers were used to assess the activity-rest pattern. Interdaily stability (IS), intradaily variability (IV), the mean value of the lowest 5 hours (L5), and the mean value of the maximum 10 hours (M10) of activity and their respective timing (TL5, TM10) were used as indicators of the activity-rest pattern throughout the day. Chronotype and social jetlag were used as indicators of circadian preference. Academic achievement, executive function, and intelligence were assessed with standardized tests. Gray matter volume was acquired by magnetic resonance imaging (MRI). IS was positively associated with executive function (β = 0.244, P = .014). IV was negatively associated with mathematics and academic applications (β: -0.211 to -0.238, P's ≤ .026). Later TM10 in the day was related to lower writing, academic skills, and intelligence (β: -0.229 to -0.271, P's ≤ .025). None of the associations found were mediated by gray matter volume. A non-fragmented and stable activity-rest pattern and earlier physical activity in the day were associated with better academic achievement, executive function, and intelligence in children with overweight/obesity. Further studies are required to corroborate or contrast our findings.

Our aim was to evaluate the circadian rhythm of motor activity, body position and integrated variable TAP (composed by wrist Temperature, motor Activity and body Position) in Sleep Disordered Breathing (SDB), its relation to SDB severity and the effect of continuous positive airway pressure (CPAP) on these circadian rhythms. To do this, we monitored motor activity and body position rhythms of 78 SDB patients (53.3 ± 1.2 years old, 26.9% women) and 32 healthy subjects (51.4 ± 3.2 years old, 43.8% women) for 1 week. On the last day of that week, SDB patients underwent a polysomnography followed by a Maintenance of Wakefulness Test, Multiple Sleep Latency Test and Sustained Attention to Response Task protocol. A subgroup of 18 moderate to severe SDB patients was treated with CPAP and monitored again after 3 months under treatment. A non-parametrical analysis was performed to characterize the circadian patterns to assess differences between groups and associations between sleep and circadian parameters. Circadian variables were altered in SDB, exhibiting a direct relationship to SDB severity. The motor activity pattern showed a clear improvement with CPAP treatment. Thus, circadian ambulatory monitoring, including the integrated variable TAP, could be used to evaluate the circadian alterations caused by SDB and activity pattern to monitor the effect of CPAP treatment.

We present evidence of pupil response modification, as well as differential theoretical melatonin suppression through selective and dynamic electrochromic filtering of visible light in the 400–500 nm range to minimize chronodisruptive nocturnal blue light exposure. A lower activation of intrinsically photosensitive retinal ganglion cells (ipRGCs), the first step for light to reach a human’s internal clock, is related to melatonin secretion therefore avoiding detrimental effects of excessive blue light exposure. Pupillary Light Reflex and Color Naming were experimentally assessed under light filtered by two different coloration states (transmissive and absorptive) of these novel dynamic filters, plus an uncoated test device, in 16 volunteers. Also, different commercial light sources at illuminances ranging from 1 to 1000 lux were differentially filtered and compared in terms of theoretical melatonin suppression. Representative parameters of the pupil responses reflected lower pupil constriction when the electrochromic filters (ECFs) were switched on (absorptive state, blue light is absorbed by the filter) compared to uncoated filters (control sample), but failed to do so under transmissive state (blue light passes through the filter) indicating less activation of ipRGCs under absorptive state (although no significant differences between states was found). Out of eight colors tested, just one showed significant differences in naming between both filter states. Thus, the ECF would have some protecting effect on ipRGC activation with very limited changes in color perception. While there are some limitations of the theoretical model used, the absorptive state yielded significantly lower theoretical melatonin suppression in all those light sources containing blue wavelengths across the illuminance range tested. This would open the way for further research on biological applications of electrochromic devices.

Background Parkinson's disease (PD) is associated with α-synuclein (αS) aggregation within the enteric nervous system (ENS) and constipation. Squalamine displaces proteins that are electrostatically bound to intracellular membranes and through this mechanism suppresses aggregation of αS monomers into neurotoxic oligomers. Objective We sought to evaluate the safety of ENT-01 oral tablets (a synthetic squalamine salt), its pharmacokinetics, and its effect on bowel function in PD patients with constipation. Methods In Stage 1, 10 patients received escalating single doses from 25 to 200 mg/day or maximum tolerated dose (MTD). In Stage 2, 34 patients received daily doses escalating from 75 to a maximum of 250 mg/day, a dose that induced change in bowel function or MTD, followed by a fixed dose for 7 days, and a 2-week washout. Primary efficacy endpoint was defined as an increase of 1 complete spontaneous bowel movement (CSBM)/week, or 3 CSBM/week over the baseline period, as defined by FDA guidelines for prokinetic agents. Safety was also assessed. Results Over 80% of patients achieved the primary efficacy endpoint, with the mean number of CSBM/week increasing from 1.2 at baseline to 3.6 during fixed dosing (p = 1.2 × 10−7). Common adverse events included nausea in 21/44 (47%) and diarrhea in 18/44 (40%) patients. Systemic absorption was Conclusions Orally administered ENT-01 was safe and significantly improved bowel function in PD, suggesting that the ENS is not irreversibly damaged in PD. Minimal systemic absorption suggests that improvements result from local stimulation of the ENS. A double-blind, placebo-controlled study is now ongoing.

Isolation from external time cues allows endogenous circadian rhythmicity to be demonstrated. In this study, also filmed as a television documentary, we assessed rhythmic changes in a healthy man time isolated in a bunker for 9 days/nights. During this period the lighting conditions were varied between: (1) self-selected light/dark cycle, (2) constant dim light, and (3) light/dark cycle with early wake up. A range of variables was assessed and related to the sleep-wake cycle, psychomotor and physical performance and clock-time estimation. This case study using modern non-invasive monitoring techniques emphasizes how different physiological circadian rhythms persist in temporal isolation under constant dim light conditions with different waveforms, free-running with a period (t) between 24 and 25 h. In addition, a significant correlation between time estimation and mid-sleep time, a proxy for circadian phase, was demonstrated.

Modern 24‐h society lifestyle is associated with experiencing frequent shifts in the lighting conditions which can negatively impact human health. Here, we use the degus, a species exhibiting diurnal and nocturnal chronotypes, to: (a) assess the impact of chronic shifts of the light:dark (LD) cycle in the animal's physiology and behaviour and (b) test the therapeutic potential of melatonin in enhancing rhythmicity under these conditions. Degus were subjected to a “5d + 2d” LD‐shifting schedule for 19 weeks. This protocol aims to mimic lighting conditions experienced by humans during shift work: LD cycle was weekly delayed by 8h during 5 “working” days (Morning, Afternoon and Night schedule); during weekends (2 days), animals were kept under Morning schedule. After 9 weeks, melatonin was provided daily for 6h in the drinking water. The “5d + 2d” shifting LD schedule led to a disruption in wheel‐running activity (WRA) and body temperature (Tb) rhythms which manifested up to three separate periods in the circadian range. This chronodisruption was more evident in nocturnal than in diurnal degus, particularly during the Afternoon schedule when a phase misalignment between WRA and Tb rhythms appeared. Melatonin treatment and, to a lesser extent, water restriction enhanced the 24‐h component, suggesting a potential role in ameliorating the disruptive effects of shift work.

During last decades, the way of life in modern societies has deeply modified the temporal adjustment of the circadian system, mainly due to the inappropriate use of artificial lighting and the high prevalence of social jet-lag. Therefore, it becomes necessary to design non-invasive and practical tools to monitor circadian marker rhythms but also its main synchronizer, the light-dark cycle under free-living conditions. The aim of this work was to improve the ambulatory circadian monitoring device (ACM, Kronowise®) capabilities by developing an algorithm that allows to determine light intensity, timing and circadian light stimulation by differentiating between full visible, infrared and circadian light, as well as to discriminate between different light sources (natural and artificial with low and high infrared composition) in subjects under free living conditions. The ACM device is provided with three light sensors: (i) a wide-spectrum sensor (380–1100 nm); (ii) an infrared sensor (700–1100 nm) and (iii) a sensor equipped with a blue filter that mimics the sensitivity curve of the melanopsin photopigment and the melatonin light suppression curve. To calibrate the ACM device, different commercial light sources and sunlight were measured at four different standardized distances with both a spectroradiometer (SPR) and the ACM device. CIE S 026/E:2018 (2018), toolbox software was used to calculate the melanopic stimulation from data recorded by SPR. Although correlation between raw data of luminance measured by ACM and SPR was strong for both full spectrum (r = 0.946, p < 0.0001) and circadian channel (r = 0.902, p < 0.0001), even stronger correlations were obtained when light sources were clustered in three groups: natural, infrared-rich artificial light and infrared-poor artificial light, and their corresponding linear correlations with SPR were considered (r = 0.997, p < 0.0001 and r = 0.998, p < 0.0001, respectively). Our results show that the ACM device provided with three light sensors and the algorithm developed here allow an accurate detection of light type, intensity and timing for full visible and circadian light, with simultaneous monitoring of several circadian marker rhythms that will open the possibility to explore light synchronization in population groups while they maintain their normal lifestyle.

An appropriate exposure to the light-dark cycle, with high irradiances during the day and darkness during the night is essential to keep our physiology on time. However, considering the increasing exposure to artificial light at night and its potential harmful effects on health (i.e. chronodisruption and associated health conditions), it is essential to understand the non-visual effects of light in humans. Melatonin suppression is considered the gold standard for nocturnal light effects, and the activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) through the assessment of pupillary light reflex (PLR) has been recently gaining attention. Also, some theoretical models for melatonin suppression and retinal photoreceptors activation have been proposed. Our aim in this study was to determine the influence of correlated color temperature (CCT) on melatonin suppression and PLR, considering two commercial light sources, as well as to explore the possible correlation between both processes. Also, the contribution of irradiance (associated to CCT) was explored through mathematical modelling on a wider range of light sources. For that, melatonin suppression and PLR were experimentally assessed on 16 healthy and young volunteers under two light conditions (warmer, CCT 3000 K; and cooler, CCT 5700 K, at ~5·1018 photons/cm2/sec). Our experimental results yielded greater post-stimulus constriction under the cooler (5700 K, 13.3 ± 1.9%) than under the warmer light (3000 K, 8.7 ± 1.2%) (p < 0.01), although no significant differences were found between both conditions in terms of melatonin suppression. Interestingly, we failed to demonstrate correlation between PLR and melatonin suppression. Although methodological limitations cannot be discarded, this could be due to the existence of different subpopulations of Type 1 ipRGCs differentially contributing to PLR and melatonin suppression, which opens the way for further research on ipRGCs projection in humans. The application of theoretical modelling suggested that CCT should not be considered separately from irradiance when designing nocturnal/diurnal illumination systems. Further experimental studies on wider ranges of CCTs and light intensities are needed to confirm these conclusions.

The present study proposes a classification model for the differential diagnosis of primary insomnia (PI) and delayed sleep phase disorder (DSPD), applying machine learning methods to circadian parameters obtained from ambulatory circadian monitoring (ACM). Nineteen healthy controls and 242 patients (PI = 184; DSPD = 58) were selected for a retrospective and non-interventional study from an anonymized Circadian Health Database (https://kronowizard.um.es/). ACM records wrist temperature (T), motor activity (A), body position (P), and environmental light exposure (L) rhythms during a whole week. Sleep was inferred from the integrated variable TAP (from temperature, activity, and position). Non-parametric analyses of TAP and estimated sleep yielded indexes of interdaily stability (IS), intradaily variability (IV), relative amplitude (RA), and a global circadian function index (CFI). Mid-sleep and mid-wake times were estimated from the central time of TAP-L5 (five consecutive hours of lowest values) and TAPM10 (10 consecutive hours of maximum values), respectively. The most discriminative parameters, determined by ANOVA, Chi-squared, and information gain criteria analysis, were employed to build a decision tree, using machine learning. This model differentiated between healthy controls, DSPD and three insomnia subgroups (compatible with onset, maintenance and mild insomnia), with accuracy, sensitivity, and AUC >85%. In conclusion, circadian parameters can be reliably and objectively used to discriminate and characterize different sleep and circadian disorders, such as DSPD and OI, which are commonly confounded, and between different subtypes of PI. Our findings highlight the importance of considering circadian rhythm assessment in sleep medicine., This work was supported by the Ministry of Economy and Competitiveness, the Instituto de Salud Carlos III through CIBERFES (CB16/10/00239) and grants 19899/GERM/15 awarded to JM and the Ministry of Science Innovation and Universities RTI2018-093528-B-I00 to MR (all of them co-financed by FEDER).

The development of wearable devices has increase interest in the use of ambulatory methods to detect sleep disorders more objectively than those permitted by subjective scales evaluating sleep quality, while subjects maintain their usual lifestyle. This study aims to validate an ambulatory circadian monitoring (ACM) device for the detection of sleep and wake states and apply it to the evaluation of sleep quality in patients with Parkinson disease (PD). A polysomnographic validation study was conducted on a group of patients with different sleep disorders in a preliminary phase, followed by a pilot study to apply this methodology to PD patients. The ACM device makes it possible to estimate the main sleep parameters very accurately, as demonstrated by: (a) the lack of significant differences between the mean values detected by PSG and ACM in time in bed (TIB), total sleep time (TST), sleep efficiency (SE), and time awake after sleep onset (WASO); (b) the slope of the correlation lines between the parameters estimated by the two procedures, very close to 1, which demonstrates the linearity of the predictions; (c) the low bias value in the estimates obtained through ACM. Sleep in PD is associated with lower distal skin temperature, efficiency and overall sleep time; greater WASO, activity during sleep and duration of naps and a worse circadian function index. In summary, the ACM device has proven to be clinically useful to evaluate sleep in an objective manner, thanks to the integrated management of different complementary variables, having advantages over conventional actigraphy.

It is widely accepted that cold exposure increases peripheral vascular resistance and arterial blood pressure (BP) and, hence, increases cardiovascular risk primarily in the elderly. However, there is a lack of concomitantly longitudinal recordings at personal level of environmental temperature (PET) and cardiophysiological variables together with skin temperatures (STs, the “interface-variable” between the body core and ambient temperature). To investigate the intra-individual temporal relationships between PET, STs and BP 60 healthy young women (52 completed the entire study) were prospectively studied in a winter/summer design for 26 h under real life conditions. The main hypothesis was tested whether distal ST (Tdist)mediates the effect of PET-changes on mean arterial BP (MAP). Diurnal profiles of cardiophysiological variables (including BP), STs and PET were ambulatory recorded. Daytime variations between 0930 and 2030 h were analyzed in detail by intra-individual longitudinal path analysis. Additionally, time segments before, during and after outdoor exposure were separately analyzed. In both seasons short-term variations in PET were positively associated with short-term changes in Tdist (not proximal ST, Tprox) and negatively with those in MAP. However, long-term seasonal differences in daytime mean levels were observed in STs but not in BP leading to non-significant inter-individual correlation between STs and BP. Additionally, higher individual body mass index (BMI) was significantly associated with lower daytime mean levels of Tprox and higher MAP suggesting Tprox as potential mediator variable for the association of BMI with MAP. In healthy young women the thermoregulatory and BP-regulatory systems are closely linked with respect to short-term, but not long-term changes in PET. One hypothetical explanation could serve recent findings that thermogenesis in brown adipose tissue is activated in a cool environment, which could be responsible for the counter-regulation of cold induced increase of BP in winter leading to no seasonal differences in MAP. Our findings suggest that the assessment of diurnal patterns of STs and PET, in addition to the conventional ambulatory BP monitoring, might improve individual cardiovascular risk prediction.

Study objectives Our aim was to evaluate the circadian rhythm of distal skin temperature (DST) in sleep-disordered breathing (SDB), its relation to excessive daytime sleepiness and the effect of continuous positive airway pressure (CPAP) on DST. Methods Eighty SDB patients (53.1 ± 1.2 years old, 27.6% women) and 67 healthy participants (52.3 ± 1.6 years old, 26.9% women) wore a temperature data logger for 1 week. On the last day of that week, SDB patients underwent a polysomnography followed by a Maintenance of Wakefulness Test (MWT), Multiple Sleep Latency Test, and Sustained Attention to Response Task protocol to objectively quantify daytime sleepiness. A subset of 21 moderate to severe SDB patients were treated with CPAP during at least 3 months and revaluated with the same procedure. A nonparametric analysis was performed to characterize DST to assess differences between groups and associations among DST, polysomnography, and daytime sleepiness measures. Results SDB patients showed an unstable, fragmented, flattened, phase-advanced, and less robust DST rhythm as compared to healthy participants. The more severe the SDB, the worse the DST pattern was, as indicated by the correlation coefficient. Sleepiness, according to MWT sleep latencies, was also associated with the higher fragmentation, lower amplitude, and less robustness of the DST rhythm. Treatment with CPAP improved DST pattern regularity and robustness. Conclusion DST is altered in SDB, exhibiting a direct relationship to the severity of this condition, and improves with CPAP treatment. DST independently correlates with sleepiness, thus, its measurement may contribute to the understanding of the pathophysiology of sleepiness in these patients.

Currently, in developed countries, nights are excessively illuminated (light at night), whereas daytime is mainly spent indoors, and thus people are exposed to much lower light intensities than under natural conditions. In spite of the positive impact of artificial light, we pay a price for the easy access to light during the night: disorganization of our circadian system or chronodisruption (CD), including perturbations in melatonin rhythm. Epidemiological studies show that CD is associated with an increased incidence of diabetes, obesity, heart disease, cognitive and affective impairment, premature aging and some types of cancer. Knowledge of retinal photoreceptors and the discovery of melanopsin in some ganglion cells demonstrate that light intensity, timing and spectrum must be considered to keep the biological clock properly entrained. Importantly, not all wavelengths of light are equally chronodisrupting. Blue light, which is particularly beneficial during the daytime, seems to be more disruptive at night, and induces the strongest melatonin inhibition. Nocturnal blue light exposure is currently increasing, due to the proliferation of energy-efficient lighting (LEDs) and electronic devices. Thus, the development of lighting systems that preserve the melatonin rhythm could reduce the health risks induced by chronodisruption. This review addresses the state of the art regarding the crosstalk between light and the circadian system. peerReviewed

Integrated multispectral devices provide convenient means for assessing the inputs to the five known photoreceptors present in the human retina. These photoreceptors drive relevant visual and non-visual pathways that control key aspects of human physiology. In this Letter we show that standard metrics of retinal photoreceptoral exposure can be quantitatively estimated, to within a 5%, by means of 12 multispectral channels, 20 nm wide (FWHM), distributed across the visible range. Full Text: PDF References D.M. Berson, F.A. Dunn, M. Takao, "Phototransduction by Retinal Ganglion Cells That Set the Circadian Clock", Science 295, 1070 (2002). CrossRef R.J. Lucas et al. "Measuring and using light in the melanopsin age", Trends in Neurosciences 37, 1 (2014). CrossRef Commission Internationale de l'Eclairage. TN 003:2015 Report on the First International Workshop on Circadian and Neurophysiological Photometry, 2013. CIE, 2015. DirectLink A. Sanchez de Miguel et al, "Evaluating Human Photoreceptoral Inputs from Night-Time Lights Using RGB Imaging Photometry", Journal of Imaging 5(4), 49 (2019). CrossRef M.S. Rea, M.G. Figueiro, A. Bierman, R. Hamner. "Modelling the spectral sensitivity of the human circadian system", Lighting Research and Technology 44, 386 (2012). CrossRef D. Cao, P.A. Barrionuevo, "Estimating photoreceptor excitations from spectral outputs of a personal light exposure measurement device", Chronobiology International 32(2), 270 (2015). CrossRef P.B. Liebelt, An Introduction to Optimal Estimation (Reading, MA, Addison-Wesley, 1967). CrossRef J. Escofet, S. Bara, "Reducing the circadian input from self-luminous devices using hardware filters and software applications", Lighting Research and Technology 49, 481 (2017). CrossRef

An integrated variable based on the combination of wrist Temperature, motor Activity and body Position (TAP) was previously developed at our laboratory to evaluate the functioning of the circadian system and sleep–wake rhythm under ambulatory conditions. However, the reliability of TAP needed to be validated with polysomnography (PSG). 22 subjects suffering from sleep disorders were monitored for one night with a temperature sensor (iButton®), an actimeter (HOBO®) and exploratory PSG. Mean waveforms, sensitivity (SE), specificity (SP), agreement rates (AR) and comparisons between TAP and sleep stages were studied. The TAP variable was optimized for SE, SP and AR with respect to each individual variable (SE: 92%; SP: 78%; AR: 86%). These results improved upon estimates previously published for actigraphy. Furthermore, TAP values tended to decrease as sleep depth increased, reaching the lowest point at phase 3. Finally, TAP estimates for sleep latency (SL: 37 ± 9 min), total sleep time (TST: 367 ± 13 min), sleep efficiency (SE: 86.8 ± 1.9%) and number of awakenings (NA > 5 min: 3.3 ± .4) were not significantly different from those obtained with PSG (SL: 29 ± 4 min; SE: 89.9 ± 1.8%; NA > 5 min: 2.3 ± .4), despite the heterogeneity of the sleep pathologies monitored. The TAP variable is a novel measurement for evaluating circadian system status and sleep–wake rhythms with a level of reliability better to that of actigraphy. Furthermore, it allows the evaluation of a patient's sleep–wake rhythm in his/her normal home environment, and at a much lower cost than PSG. Future studies in specific pathologies would verify the relevance of TAP in those conditions.

Introduction. Patients with dementia, especially Alzheimer’s disease, present several circadian impairments related to an accelerated perturbation of their biological clock that is caused by the illness itself and not merely age-related. Thus, the objective of this work was to elucidate whether these circadian system alterations were already present in patients with mild cognitive impairment (MCI), as compared to healthy age-matched subjects.Methods. 40 subjects (21 patients diagnosed with MCI, 74.1 ± 1.5 y.o., and 19 healthy subjects, 71.7 ± 1.4 y.o.) were subjected to ambulatory monitoring, recording wrist skin temperature, motor activity, body position, and the integrated variable TAP (including temperature, activity, and position) for one week. Nonparametrical analyses were then applied.Results. MCI patients exhibited a significant phase advance with respect to the healthy group for the following phase markers: temperature M5 (mean ± SEM: 04:20 ± 00:21 versus 02:52 ± 00:21) and L10 (14:35 ± 00:27 versus 13:24 ± 00:16) and TAP L5 (04:18 ± 00:14 versus 02:55 ± 00:30) and M10 (14:30 ± 00:18 versus 13:28 ± 00:23).Conclusions. These results suggest that significant advances in the biological clock begin to occur in MCI patients, evidenced by an accelerated aging of the circadian clock, as compared to a healthy population of the same age.

Modern societies are characterized by a 24/7 lifestyle (LS) with no environmental differences between day and night, resulting in weak zeitgebers (weak day light, absence of darkness during night, constant environmental temperature, sedentary LS and frequent snacking), and as a consequence, in an impaired circadian system (CS) through a process known as chronodisruption. Both weak zeitgebers and CS impairment are related to human pathologies (certain cancers, metabolic syndrome and affective and cognitive disorders), but little is known about how to chronoenhance the CS. The aim of this work is to propose practical strategies for chronoenhancement, based on accentuating the day/night contrast. For this, 131 young subjects were recruited, and their wrist temperature (WT), activity, body position, light exposure, environmental temperature and sleep were recorded under free-living conditions for 1 week. Subjects with high contrast (HC) and low contrast (LC) for each variable were selected to analyze the HC effect in activity, body position, environmental temperature, light exposure and sleep would have on WT. We found that HC showed better rhythms than LC for every variable except sleep. Subjects with HC and LC for WT also demonstrated differences in LS, where HC subjects had a slightly advanced night phase onset and a general increase in day/night contrast. In addition, theoretical high day/night contrast calculated using mathematical models suggests an improvement by means of LS contrast. Finally, some individuals classified as belonging to the HC group in terms of WT when they are exposed to the LS characteristic of the LC group, while others exhibit WT arrhythmicity despite their good LS habits, revealing two different WT components: an exogenous component modified by LS and another endogenous component that is refractory to it. Therefore, intensifying day/night contrast in subject's LS has proven to be a feasible measure to chronoenhance the CS.

The robustness of the circadian timing system (CTS) was correlated to quality of life and predicted for improved survival in cancer patients. However, chemotherapy disrupted the CTS according to dose and circadian timing in mice. A continuous and repeated measures longitudinal design was implemented here to characterize CTS dynamics in patients receiving a fixed circadian-based chemotherapy protocol. The rest-activity rhythm of 49 patients with advanced cancer was monitored using a wrist actigraph for 13 days split into four consecutive spans of 3-4 days each, i.e., before, during, right after and late after a fixed chronotherapy course. The relative amount of activity in bed vs. out of bed (I

Melatonin is a pleiotropic molecule with many cellular and systemic actions, including chronobiotic effects. Beneficial effects are widely documented concerning the treatment of neoplastic diseases in vivo as well as reductions in viability of cultured cells from melanoma, one of the most aggressive cancers in humans. However, studies of its effects on non-tumor cells in vitro have not focused on viability, except for experiments aiming to protect against oxidotoxicity or other toxicological insults. Furthermore, there is no agreement on the range of effective melatonin concentrations in vitro, and the mechanisms that reduce cell viability have remained unclear. Tumor cell-specific increases in the production of reactive oxygen and nitrogen species (ROS/RNS) may provide a possible explanation. Our aim was to analyze the potential inhibition of tumor (B16 melanoma 4A5) and non-tumor cell (3T3 Swiss albino) viability using a wide range of melatonin concentrations (10-11-10-2 M), and to determine whether intracellular ROS enhancement was involved in this process. In the absence of fetal bovine serum (FBS), low melatonin concentrations (10-9-10-5 M) reduced the proliferation of melanoma cells with no effect in fibroblasts, whereas, in the presence of FBS, they had no effect or even increased the proliferation of both fibroblast and melanoma cells. Melatonin concentrations in the upper millimolar range increased ROS levels and reduced the viability of both cell types, but more markedly so in non-tumor cells. Thus, low melatonin concentrations reduce proliferation in this specific melanoma cell line, whereas high concentrations affect the viability of both tumor (B16 4A5 melanoma) and non-tumor (3T3 fibroblasts) cells. Increased ROS levels in both lines indicate a role for ROS production in the reduction of cell viability at high-but not low-melatonin concentrations, although the mechanism of action still remains to be elucidated. © 2013 by the authors; licensee MDPI, Basel, Switzerland., This project was funded by the Instituto de Salud Carlos III (RETICEF, RD06/0013/0019; RD06/0013/0001 and RD12/0043/0011), the Ministry of Education and Science (BFU2010-21945-C02-01) and a Research fellowship granted to MA Bonmatí (AP2009-1051).

Intrinsically photosensitive retinal ganglion cells (ipRGCs), whose photopigment melanopsin has a peak of sensitivity in the short wavelength range of the spectrum, constitute a common light input pathway to the olivary pretectal nucleus (OPN), the pupillary light reflex (PLR) regulatory centre, and to the suprachiasmatic nuclei (SCN), the major pacemaker of the circadian system. Thus, evaluating PLR under short wavelength light (λmax ≤ 500 nm) and creating an integrated PLR parameter, as a possible tool to indirectly assess the status of the circadian system, becomes of interest. Nine monochromatic, photon-matched light stimuli (300 s), in 10 nm increments from λmax 420 to 500 nm were administered to 15 healthy young participants (8 females), analyzing: i) the PLR; ii) wrist temperature (WT) and motor activity rhythms (WA), iii) light exposure (L) pattern and iv) diurnal preference (Horne-Östberg), sleep quality (Pittsburgh) and daytime sleepiness (Epworth). Linear correlations between the different PLR parameters and circadian status index obtained from WT, WA and L recordings and scores from questionnaires were calculated. In summary, we found markers of robust circadian rhythms, namely high stability, reduced fragmentation, high amplitude, phase advance and low internal desynchronization, were correlated with a reduced PLR to 460-490 nm wavelengths. Integrated circadian (CSI) and PLR (cp-PLR) parameters are proposed, that also showed an inverse correlation. These results demonstrate, for the first time, the existence of a close relationship between the circadian system robustness and the pupillary reflex response, two non-visual functions primarily under melanopsin-ipRGC input.

Alzheimer disease (AD) is a neurodegenerative disorder that primarily causes β-amyloid accumulation in the brain, resulting in cognitive and behavioral deficits. AD patients, however, also suffer from severe circadian rhythm disruptions, and the underlying causes are still not fully known. Patients with AD show reduced systemic melatonin levels. This may contribute to their symptoms, since melatonin is an effective chronobiotic and antioxidant with neuroprotective properties. Here, the authors critically assessed the effects of long-term melatonin treatment on circadian system function, hippocampal oxidative stress, and spatial memory performance in the APPswe/PS1 double transgenic (Tg) mouse model of AD. To test if melatonin MT1/MT2 receptor activation, alone, was involved, the authors chronically treated some mice with the selective MT1/MT2 receptor agonist ramelteon. The results indicate that many of the circadian and behavioral parameters measured, including oxidative stress markers, were not significantly affected in these AD mice. During the day, though, Tg controls (Tg-CON) showed significantly higher mean activity and body temperature (BT) than wild-type (WT) mice. Overall, BT rhythm amplitude was significantly lower in Tg than in WT mice. Although melatonin treatment had no effect, ramelteon significantly reduced the amplitude of the BT rhythm in Tg mice. Towards the end of the experiment, Tg mice treated with ramelteon (Tg-RAM) showed significantly higher circadian rhythm fragmentation than Tg-CON and reduced circadian BT rhythm strength. The free-running period (τ) for the BT and locomotor activity (LA) rhythms of Tg-CON was24 h. Whereas melatonin maintained τ at 24 h for BT and LA in both genotypes, ramelteon treatment had no effect. In the behavioral tests, the number of approaches and time spent exploring novel objects were significantly higher in Tg-CON than WT controls. Brain tissue analysis revealed significant reduction in hippocampal protein oxidation in Tg-MEL and Tg-RAM compared with Tg-CON animals. These results suggest that not all aspects of the circadian system are affected in the APPswe/PS1 mice. Therefore, care should be taken when extending the results obtained in Tg mice to develop new therapies in humans. This study also revealed the complexity in the therapeutic actions of melatonin and ramelteon in this mouse model of AD.

Octodon degus is a primarily diurnal rodent that presents great variation in its circadian chronotypes due to the interaction between two phase angles of entrainment, diurnal and nocturnal, and the graded masking effects of environmental light and temperature. The aim of this study was to test whether the circadian system of this diurnal rodent can be internally dissociated by imposing cycles shorter and longer than 24 h, and to determine the influence of degus chronotypes and wheel-running availability on such dissociation. To this end, wheel-running activity and body temperature rhythms were studied in degus subjected to symmetrical light-dark (LD) cycles of T28h and T21h. The results show that both T-cycles dissociate the degus circadian system in two different components: one light-dependent component (LDC) that is influenced by the presence of light, and a second non-light-dependent component (NLDC) that free-runs with a period different from the external lighting cycle. The LDC was more evident in the nocturnal than diurnal chronotype, and also when wheel running was available. Our results show that, in addition to rats and mice, degus must be added to the list of species that show an internal dissociation in their circadian rhythms when exposed to forced desynchronization protocols. The existence of a multioscillatory circadian system having two groups of oscillators with low coupling strength may explain the flexibility of degus chronotypes.

Octodon degus, a mainly diurnal rodent, characterized by its ability to shift to a nocturnal locomotor pattern under laboratory conditions, was studied to determine whether restricted food access during the scotophase could induce nocturnalism. To address this question, wheel running activity, feeding, and body temperature rhythms were analyzed for diurnal degus housed with a wheel and subjected to either long (12 h) or short (2 h) food availability periods, in the latter case with random or scheduled food access times. The results show that allowing nocturnal feeding for 2 h, but not 12 h, can shift a previous diurnal phase preference for wheel running activity and body temperature to the scotophase, with random feeding being more effective than scheduled food availability. However, this behavioral inversion proved to be unstable, as the degus returned to the diurnal phase within only a few days after the restricted feeding was discontinued. In addition, the negative masking effect induced by light, which is characteristic of the degus' nocturnal chronotype, was not observed when the animals were forced to feed at night. Thus, neither long, short, random, nor scheduled food-availability during the scotophase was able to induce all the characteristics of the nocturnal chronotype in Octodon degus.

Animal models of anxiety have usually employed nocturnal species (e.g. rats and mice), and the tests used have been almost exclusively performed during the diurnal phase (lights on). Here, for the first time, we tested anxiety in a diurnal rodent, Octodon degus, according to its age (23 versus 40 months of age) and the time of the day. We employed three anxiety tests: object recognition, open field and light-dark tests, which were applied in the morning, at mid-day and in the late afternoon, respectively. Adult animals spent more time exploring a new object than aged animals. Nevertheless, there were no differences in the frequency of object exploration or in the latency to the first object approach between the groups. In the open field test, adult animals spent more time exploring (ambulation and rearing) than aged ones. Although both groups exhibited similar frequencies in transition from the dark to the light box in the light-dark test, adult animals spent significantly more time in the light, and expressed less anxiety when making the decision to cross over from the dark into the light area. In conclusion, there were no differences in anxiety between adult and aged animals in the morning session, although adult animals were more attentive when exploring a new object. However, in the mid-day and afternoon testing sessions, aged animals were more anxious than adults.

Processes involved in the operation of the circadian pacemaker are well characterized; however; little is known about what mechanisms drive the overt diurnal, nocturnal, or crepuscular behavior in a species. In this context, dual-phasing rodents, such as Octodon degus, emerge as a useful model to decipher these keys. Two main chronotypes, nocturnal and diurnal, have been traditionally described in laboratory-housed degus based on the percentage of activity displayed by the animals during the scotophase or photophase. However, if one considers also the entrainment phase angle during the first days following a change from LD to DD conditions, a third chronotype (intermediate)-or more properly, a continuous grading of circadian expressions between diurnal and nocturnal chronotype-can be observed. Our experiments suggest the pacemaker of the diurnal animal is entrained to the photophase, and light does not exert a negative masking effect. The pacemaker of the nocturnal degus, on the other hand, is entrained to the scotophase, and light exerts a strong negative masking effect. Finally, the intermediate chronotype is characterized by variable negative masking effect of light overlapping a pacemaker entrained to the photophase. The phase shift inversion from diurnal to nocturnal chronotype is related to the availability of a wheel in the cage, and the effect may be located downstream from the clock. However, body temperature rhythm recordings, less affected by masking effects, point to an involvement of the circadian pacemaker in chronotype differentiation, as transient entrainment cycles, and not an abrupt phase shift, were detected after providing access to the wheel. The diurnality of degus seems to be the result of a variety of mechanisms, which may explain how different processes can lead to similar chronotypes.

Chronobiology is the scientific discipline that deals with the study of the biological rhythms and their underlying mechanisms. The alteration of biological rhythms, such as blood pressure or temperature begins to be considered as a good marker of certain diseases and senescence. Among the variables, the wrist skin temperature has proven to be a good marker of the circadian rhythms of the subject. In this paper we evaluate the wrist temperature of four groups of subjects with different age in order to gain some knowledge on the evolution of the circadian rhythms and its application to age classification.

Background:\ud Adequate circadian timing of cancer treatment schedules (chronotherapy) can enhance tolerance and efficacy several-fold in experimental and clinical situations. However, the optimal timing varies according to sex, genetic background and lifestyle. Here, we compute the individual phase of the Circadian Timing System to decipher the internal timing of each patient and find the optimal treatment timing.\ud \ud Methods:\ud Twenty-four patients (11 male; 13 female), aged 36 to 77 years, with advanced or metastatic gastro-intestinal cancer were recruited. Inner wrist surface Temperature, arm Activity and Position (TAP) were recorded every 10 min for 12 days, divided into three 4-day spans before, during and after a course of a set chronotherapy schedule. Pertinent indexes, I

The confinement of critically ill patients in intensive care units (ICU) imposes environmental constancy throughout both day and night (continuous light, noise, caring activities medications, etc.), which has a negative impact on human health by inducing a new syndrome known as circadian misalignment, circadian disruption or chronodisruption (CD). This syndrome contributes to poor sleep quality and delirium, and may impair septic states frequently observed in critically ill patients. However, and although the bidirectional crosstalk between CD with sleep impairment, delirium and inflammation in animal models has been known for years and has been suspected in ICU patients, few changes have been introduced in the environment and management of ICU patients to improve their circadian rhythmicity. Delirium, the most serious condition because it has a severe effect on prognosis and increases mortality, as well as sleep impairment and sepsis, all three of them linked to disorganization of the circadian system in critically ill patients, will be revised considering the functional organization of the circadian system, the main input and output signals that synchronize the clock, including a brief description of the molecular circadian clock machinery, the non-visual effects of light, and the ICU light environment. Finally, the potential usefulness of increased light/dark contrast and melatonin treatment in this context will be analyzed, including some practical countermeasures to minimize circadian disruption and improve circadian system chronoenhancement, helping to make these units optimal healing environments for patients.

In circadian terms, human ontogeny is characterized by the emergence of a daily pattern, from a previous ultradian pattern, for most variables during the first 6 months of life. Circadian aging in humans is characterized by a phase advance, accompanied by rhythm fragmentation and flattening. Despite an expanding body of literature focused on distal skin temperature, little information is available about the ontogeny and practically nothing about age-related changes in this rhythm. Thus, the aim was to evaluate the degree of maturation and aging of the circadian pattern of distal skin temperature to identify those parameters that are modified throughout life and could be used to differentiate subjects according to their age. For this, distal skin temperature was measured in 197 volunteers (55 % women), including babies aged 15 days (30 subjects), 1 month (28 subjects), 3 months (31 subjects), and 6 months (10 subjects); young adults aged 19 years (37 subjects); middle-aged persons aged 46 years (27 subjects); older people aged 72 (34 subjects). Circadian system maturation was associated with an increase in amplitude and a reduction in skin temperature during sleep. During adulthood, women showed a more robust pattern (lower fragmentation, and higher night-time temperature, amplitude, circadian function index, and first harmonic relative power); however, these differences were lost with aging, a period of life that was consistently associated with a phase advance of the rhythm. In summary, distal skin temperature pattern can be used as a robust variable to discern between different ages throughout the life.

The robustness of the circadian timing system (CTS) was correlated to quality of life and predicted for improved survival in cancer patients. However, chemotherapy disrupted the CTS according to dose and circadian timing in mice. A continuous and repeated measures longitudinal design was implemented here to characterize CTS dynamics in patients receiving a fixed circadian-based chemotherapy protocol. The rest-activity rhythm of 49 patients with advanced cancer was monitored using a wrist actigraph for 13 days split into four consecutive spans of 3-4 days each, i.e., before, during, right after and late after a fixed chronotherapy course. The relative amount of activity in bed vs. out of bed (IO, main endpoint), the autocorrelation coefficient r24, the relative 24-hr amplitude (Amp), interdaily stability (IS) and intradaily variability (IV) were compared according to study span. Circadian disruption (IO ≤ 97.5%) resulted from the administration of the fixed chronotherapy protocols, with all five rest-activity rhythm parameters being worsened in the whole group of patients (p0.05). Mean parameter values subsequently recovered to near baseline values. The occurrence of circadian disruption on chemotherapy was associated with a higher risk of clinically relevant fatigue (p = 0.028) or body weight loss (p = 0.05). Four CTS dynamic patterns characterized treatment response including no change (9.5% of the patients); improvement (14.3%); alteration and complete recovery (31%) or sustained deterioration (45%), possibly due to inadequate chronotherapy dosing and/or timing. Improved clinical tolerability could result from the minimization of circadian disruption through the personalization of chronotherapy delivery.

Clock gene expression is not only confined to the master circadian clock in the suprachiasmatic nucleus (SCN) but is also found in many other brain regions. The phase relationship between SCN and extra-SCN oscillators may contribute to known differences in chronotypes. The Octodon degus is a diurnal rodent that can shift its activity-phase preference from diurnal to nocturnal when running wheels become available. To understand better the relationship between brain clock gene activity and chronotype, we studied the day-night expression of the Period genes, Per1 and Per2, in the SCN and extra-SCN brain areas in diurnal and nocturnal degus. Since negative masking to light and entrainment to the dark phase are involved in the nocturnalism of this species, we also compare, for the first time, Per expression between entrained (EN) and masked nocturnal (MN) degus. The brains of diurnal, MN, and EN degus housed with wheels were collected during the light (ZT4) and dark (ZT16) phases. Per1 and Per2 mRNA levels were analyzed by in situ hybridization. Within the SCN, signals for Per1 and Per2 were higher at ZT4 irrespective of chronotype. However, outside of the SCN, Per1 expression in the hippocampus of EN degus was out of phase (higher values at ZT16) with SCN values. Although a similar trend was seen in MN animals, this day-night difference in Per1 expression was not significant. Interestingly, daily differences in Per1 expression were not seen in the hippocampus of diurnal degus. For other putative brain areas analyzed (cortices, striatum, arcuate, ventromedial hypothalamus), no differences in Per1 levels were found between chronotypes. Both in diurnal and nocturnal degus, Per2 levels in the hippocampus and in the cingulate and piriform cortices were in phase with their activity rhythms. Thus, diurnal degus showed higher Per2 levels at ZT4, whereas in both types of nocturnal degus, Per2 expression was reversed, peaking at ZT16. Together, the present study supports the hypothesis that the mechanisms underlying activity-phase preference in diurnal and nocturnal mammals reside downstream from the SCN, but our data also indicate that there are fundamental differences between nocturnal masked and entrained degus.

Aims To determine how prevalent circadian rhythm impairments are in nurses working in medical, surgical and intensive care units in five Spanish hospitals and how the quality of night-time sleeping and sleepiness affect the nurses′ morning and evening chronotypes. Background Shift work is a recognized work pattern for nurses in all countries. Given the important role that nurses play in hospital care, it is vital to establish what repercussions this has on the nurses′ working schedules and how any disturbance in circadian rhythm affects patient safety. Design A multicentre, observational, descriptive and cross-sectional study in seven hospitals in the Spanish National Health System. Method A stratified sample of 1,300 nurses is being collected in three types of units: medical, surgical and intensive care. The 3-year study started in January 2012 and will continue until December 2014, with no exclusion criteria. The Kronowise® will be used to monitor the nurses' circadian rhythms, by recording their activity, position and wrist temperature. We will also use three questionnaires to evaluate sleep quality, daytime drowsiness and chronotype: (a) Pittsburg Sleep Quality Index; (b) Epworth Daytime Sleepiness Scale; and (c) Morning and Evening Questionnaire. Data will be collected from each hospital and statistical analysis will be carried out using the SPSS 19.0. Discussion The study findings will show the current state of the nurses' circadian rhythms and how shift work can affect them and their job performance. Funding for this 3-year study was granted in December 2011 by the Spanish Health Research Fund (PI 11/00646, Health Ministry). This project is also funded by the Instituto de Salud Carlos III (RETICEF, RD12/0043/0011, RD12/0043/0006).

Social isolation in adulthood is a psychosocial stressor that can result in endocrinological and behavioral alterations in different species. In rodents, controversial results have been obtained in fear conditioning after social isolation at adulthood, while neural substrates underlying these differences are largely unknown. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) are prominent modulators of synaptic plasticity underlying memory processes in many tasks, including fear conditioning. In this study, we used adult female Octodon degus to investigate the effects of long-term social isolation on contextual and cued fear conditioning, and the possible modulation of the synaptic levels of NCAM and PSA-NCAM in the hippocampus. After 6½ months of social isolation, adult female degus showed a normal auditory-cued fear memory, but a deficit in contextual fear memory, a hippocampal dependent task. Subsequently, we observed reduced hippocampal synaptic levels of PSA-NCAM in isolated compared to grouped-housed female degus. No significant differences were found between experimental groups in hippocampal levels of the three main isoforms of NCAM (NCAM180, NCAM140 and NCAM120). Interestingly, social isolation reduced the volume of the hippocampal CA1 subfield, without affecting the volume of the CA3 subregion or the total hippocampus. Moreover, attenuated body weight gain and reduced number of granulocytes were detected in isolated animals. Our findings indicate for the first time, that long-term social isolation of adult female animals induces a specific shrinkage of CA1 and a decrease in synaptic levels of PSA-NCAM in the hippocampus. These effects may be related to the deficit in contextual fear memory observed in isolated female degus.

Daily exposure to environmental light is the most important zeitgeber in humans, and all studied characteristics of light pattern (timing, intensity, rate of change, duration, and spectrum) influence the circadian system. However, and due to lack of current studies on environmental light exposure and its influence on the circadian system, the aim of this work is to determine the characteristics of a naturalistic regimen of light exposure and its relationship with the functioning of the human circadian system. Eighty-eight undergraduate students (18-23 yrs) were recruited in Murcia, Spain (latitude 38°01'N) to record wrist temperature (WT), light exposure, and sleep for 1 wk under free-living conditions. Light-exposure timing, rate of change, regularity, intensity, and contrast were calculated, and their effects on the sleep pattern and WT rhythm were then analyzed. In general, higher values for interdaily stability, relative amplitude, mean morning light, and light quality index (LQI) correlated with higher interdaily stability and relative amplitude, and phase advance in sleep plus greater stability in WT and phase advance of the WT circadian rhythm. On the other hand, a higher fragmentation of the light-exposure rhythm was associated with more fragmented sleep. Naturalistic studies using 24-h ambulatory light monitoring provide essential information about the main circadian system input, necessary for maintaining healthy circadian tuning. Correcting light-exposure patterns accordingly may help prevent or even reverse health problems associated with circadian disruption.

The authors developed a method useful for home measurement of temperature, activity, and sleep rhythms in infants under normal-living conditions during their first 6 mos of life. In addition, parametric and nonparametric tests for assessing circadian system maturation in these infants were compared. Anthropometric parameters plus ankle skin temperature and activity were evaluated in 10 infants by means of two data loggers, Termochron iButton (DS1291H, Maxim Integrated Products, Sunnyvale, CA) for temperature and HOBO Pendant G (Hobo Pendant G Acceleration, UA-004-64, Onset Computer Corporation, Bourne, MA) for motor activity, located in special baby socks specifically designed for the study. Skin temperature and motor activity were recorded over 3 consecutive days at 15 days, 1, 3, and 6 mos of age. Circadian rhythms of skin temperature and motor activity appeared at 3 mos in most babies. Mean skin temperature decreased significantly by 3 mos of life relative to previous measurements (p = .0001), whereas mean activity continued to increase during the first 6 mos. For most of the parameters analyzed, statistically significant changes occurred at 3-6 mos relative to 0.5-1 mo of age. Major differences were found using nonparametric tests. Intradaily variability in motor activity decreased significantly at 6 mos of age relative to previous measurements, and followed a similar trend for temperature; interdaily stability increased significantly at 6 mos of age relative to previous measurements for both variables; relative amplitude increased significantly at 6 mos for temperature and at 3 mos for activity, both with respect to previous measurements. A high degree of correlation was found between chronobiological parametric and nonparametric tests for mean and mesor and also for relative amplitude versus the cosinor-derived amplitude. However, the correlation between parametric and nonparametric equivalent indices (acrophase and midpoint of M5, interdaily stability and Rayleigh test, or intradaily variability and P(1)/P(ultradian)) despite being significant, was lower for both temperature and activity. The circadian function index (CFI index), based on the integrated variable temperature-activity, increased gradually with age and was statistically significant at 6 mos of age. At 6 mos, 90% of the infants' rest period coincided with the standard sleep period of their parents, defined from 23:00 to 07:00 h (dichotomic index I O; when I O = 100%, there is a complete coincidence between infant nocturnal rest period and the standard rest period), whereas at 15 days of life the coincidence was only 75%. The combination of thermometry and actimetry using data loggers placed in infants' socks is a reliable method for assessing both variables and also sleep rhythms in infants under ambulatory conditions, with minimal disturbance. Using this methodological approach, circadian rhythms of skin temperature and motor activity appeared by 3 mos in most babies. Nonparametric tests provided more reliable information than cosinor analysis for circadian rhythm assessment in infants.

This work was supported by MICINN (AP2006-04117, BFU2007-60658/BFI, and BFU2009-07793/BFI), Instituto de Salud Carlos III (RETICS RD07/0062/0012, RETICEF RD06/0013/0019), FUNDALUCE, ONCE, Fundacion Medica Mutua Madrilena.

Daily rhythms in different biochemical and hematological variables have been widely described in either diurnal or nocturnal species, but so far no studies in the rhythms of these variables have been conducted in a dual-phasing species such as the degus. The Octodon degus is a rodent that has the ability to switch from diurnal to nocturnal activity under laboratory conditions in response to wheel-running availability. This species may help us discover whether a complete temporal order inversion occurs parallel to the inversion that has been observed in this rodent's activity pattern. The aim of the present study is to determine the phase relationships among 26 variables, including behavioral, physiological, biochemical, and hematological variables, during the day and at night, in diurnal and nocturnal degus chronotypes induced under controlled laboratory conditions through the availability of wheel running. A total of 39 male degus were individually housed under a 12:12 light-dark (LD) cycle, with free wheel-running access. Wheel-running activity (WRA) and body temperature (Tb) rhythms were recorded throughout the experiment. Melatonin, hematological, and biochemical variables were determined by means of blood samples obtained every 6 h (ZT1, ZT7, ZT13, and ZT19). In spite of great differences in WRA and Tb rhythms between nocturnal and diurnal degus, no such differences were observed in the temporal patterns of most of the biological variables analyzed for the two chronotypes. Variation was only found in plasma urea level and lymphocyte number. A slight delay in the phase of the melatonin rhythm was also observed. This study shows the internal temporal order of a dual-phasing mammal does not show a complete inversion in accordance with its activity and body temperature pattern; it would appear that the switching mechanism involved in the degu's nocturnalism is located downstream from the pacemaker.