Abolished circadian rhythm of salivary cortisol in elite artistic gymnasts

Objective

The aim of this study was to evaluate the effects of intensive physical exercise and acute psychological stress during high level athletic competition as reflected on the levels of salivary cortisol in elite artistic gymnasts (AGs).

Design

The study included 239 AGs (142 females-97 males) who participated in the European Championship of Gymnastics in 2006 and 81 adolescents (40 females-41 males), matched for age, as controls. All athletes participated voluntarily in all or parts of the study, providing samples or data for each of the variables measured. Height, weight, body fat, lean body mass (LBM), bone age and Tanner stage of puberty were assessed and data concerning the time of thelarche, adrenarche and menarche as well as, the onset and the intensity (hours per week) of training were obtained.

Methods

Saliva samples were collected, the morning before training and in the afternoon shortly after the competition. From controls, the saliva samples were collected in the morning. Cortisol concentrations were measured using a chemiluminescence method. Acute stress was assessed using a questionnaire designed for the study.

Results

No difference was found between morning and afternoon salivary cortisol levels in both male and female AGs (females: AM: 15.45 ± 7.45 nmol/l vs PM: 15.73 ± 9.38 nmol/l; males: AM: 10.21 ± 5.52 nmol/l vs PM: 9.93 ± 13.8 nmol/l, p > 0.05).Female AGs presented higher levels of morning salivary cortisol than female controls (p < 0.05). Both male and female AGs had higher degree of psychological stress in comparison with controls (p < 0.001, p < 0.013, respectively). Female AGs had higher morning and afternoon salivary cortisol levels (p < 0.01, p < 0.01, respectively) and higher degree of stress (p < 0.003) than males.

Conclusions

In elite AGs the diurnal rhythm of salivary cortisol has been abolished, probably due to the strenuous training and competition conditions. Female AGs presented higher levels of morning salivary cortisol and psychological stress compared to both male AGs and female controls. The long term consequences of these modifications of the HPA axis remain to be elucidated.     

Glucocorticoid response to exercise as measured by serum and salivary cortisol

Serum and salivary cortisol concentrations were studied in 78 elite athletes engaged in different sports, by subjecting them to high-intensity laboratory exercise. The mean difference in the pre-exercise cortisol concentrations in the seven groups studied were more marked in serum (from 311 to 768 nmol.l-1) than in saliva (from 17.9 to 22.7 nmol.l-1, only one group reaching 40 nmol.l-1). Judging from the correlation coefficients based on total variances, the post-/pre-exercise differences in cortisol concentrations in serum depended chiefly on pre-exercise values, while those in saliva tended to depend more on the postexercise concentrations. The coefficients of correlation between that difference and either the pre- or postexercise values were -0.71 and 0.47, respectively, for serum, and -0.51 and 0.58, respectively, for saliva. This would suggest that salivary cortisol concentration might be a more suitable variable for assessing glucocorticoid activity in exercise than serum cortisol concentration, probably being less sensitive to pre-exercise emotional state.     

Bacteria in the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

Bacteria load in saliva was experimentally manipulated, and the consequences for the measurement of salivary testosterone (T), dehydroepiandrosterone (DHEA), and cortisol (C) were examined. Healthy adults (n = 19) donated the first saliva sample upon rising after which they rinsed their mouths with water, waited 10 min, and donated a second sample. Samples were either left untreated or passed through a 0.22-microm filter and then frozen at -80 degrees C or incubated at room temperature (RT) for 10 days. Aliquots of each sample were cultured on agar to determine baseline and post-incubation (or freezing) bacteria load. Bacteria counts were not significantly influenced by rinsing (with water), were substantially reduced by filtration, and increased by incubation at RT. Average levels of salivary T and C, but not DHEA, were significantly lower in samples stored at RT than samples frozen the day of collection. The change in bacteria count induced by storing samples at RT was associated with a change in testosterone but not cortisol or DHEA. When samples were passed through a 0.22-microm filter bacteria counts were reduced, and the association between bacteria and testosterone was reduced to non-significant. These findings contribute to a growing body of literature revealing that the process of sample collection, storage, and handling can dramatically influence the accuracy of information generated when salivary biomarkers are integrated into research and clinical diagnostics.     

Assessment of adrenal function by measurement of salivary steroids in response to corticotrophin in patients infected with human immunodeficiency virus

OBJECTIVE: Adrenal insufficiency has been reported among critically ill HIV-infected patients. This is the first study that attempts to detect subclinical hypoadrenal states in non-critical HIV patients through salivary steroids in response to intramuscular low-dose ACTH injection. PATIENTS AND METHODS: We studied 21 ambulatory adult HIV-infected patients without specific clinical signs or symptoms of adrenal insufficiency. Normal salivary flow-rate and salivary alpha-amylase activity confirmed adequate salivary gland function. Salivary cortisol (SAF) and salivary aldosterone (SAL) were obtained at baseline and 30 min after the injection of 25 microg of ACTH in the deltoid muscle (LDT(s)). Assessment of salivary steroids after stimulation with 250 microg of intramuscular ACTH (HDT(s)) was performed on those who hyporesponded to LDT(s). Basal blood samples were drawn for steroids, renin and ACTH measurements. RESULTS: At baseline SAF and SAL correlated significantly (p=0.0001) with basal serum cortisol and aldosterone (r=0.70 and 0.91, respectively). Plasma ACTH and renin concentrations were within the normal range in all patients. Eight of the twenty-one HIV(+) patients were LDT(s) hyporesponders in either SAF (n:1) or SAL (n:7). LDT(s) repeated in six cases after a year reconfirmed the impairment of aldosterone secretion. LDT(s) hyporesponders had normal steroid responses to HDT(s). CONCLUSIONS: LDT(s) is a simple, safe, well-accepted and non-invasive approach to assess adrenal function in HIV-infected ambulatory patients. It revealed subnormal cortisol (5%) and aldosterone responses (33%) when HDT(s) results were normal.     

Permeability of submandibular salivary glands in dogs to blood-borne horseradish peroxidase (HRP)

Summary Horseradish peroxidase (HRP) was administered to the submandibular glands of dogs by close-arterial bolus-type injections, and its localisation was examined histochemically by light and electron microscopy. The HRP became widespread in the interstices of the glands and reached many central acinar lumina via scattered localised parts of their tight junctional complexes. Reaction product was less often found in the lumina of demilunes, which suggested that the intercellular junctions there were less leaky. HRP was often found in sizeable spaces between myoepithelial cells and the underlying parenchymal cells; such large spaces have not been observed in this situation in other species. The possibility that permeability pathways may arise intermittently at different sites in the adhering mechanisms between the acinar cells is discussed.It is concluded that potential paracellular permeability pathways for macromolecules exist in these glands and, if the concentration gradient is sufficiently high, molecules even as large as those of HRP can to some extent permeate passively from the interstices to the saliva. In resting glands the principal permeability site is between the central acinar cells.Supported by Grants from the M.R.C. and the V.R.T. King's College HospitalWe wish to acknowledge the technical help of Mr. K.J. Davies and Mr. P.S.A. Rowley     

Controlled exposure to light and darkness realigns the salivary cortisol rhythm in night shift workers

The efficacy of a light/darkness intervention designed to promote circadian adaptation to night shift work was tested in this combined field and laboratory study. Six full-time night shift workers (mean age ± SD:37.1 ± 8.1 yrs) were provided an intervention consisting of an intermittent exposure to full-spectrum bright white light (∼2000 lux) in the first 6 h of their 8 h shift, shielding from morning light by tinted lenses (neutral gray density, 15% visual light transmission), and regular sleep/darkness episodes in darkened quarters beginning 2 h after the end of each shift. Five control group workers (41.1 ± 9.9 yrs) were observed in the presence of a regular sleep/darkness schedule only. Constant routines (CR) performed before and after a sequence of ∼12 night shifts over 3 weeks revealed that treatment group workers displayed significant shifts in the time of peak cortisol expression and realignment of the rhythm with the night-oriented schedule. Smaller phase shifts, suggesting an incomplete adaptation to the shift work schedule, were observed in the control group. Our observations support the careful control of the pattern of light and darkness exposure for the adaptation of physiological rhythms to night shift work.     

A note on the effectiveness of behavioural rehabilitation for reducing inter-dog aggression in shelter dogs

The effectiveness of a rehabilitation program for reducing inter-dog aggression was evaluated at the municipal animal shelter. Sixteen dogs (of 60 examined) met the study criteria of medium inter-dog aggression as determined by an inter-dog aggression test. These dogs received a 10-day treatment of daily rehabilitation for 30 min (rehabilitation group, n = 9) or daily release into an outdoor enclosure for 30 min (control group, n = 7). Rehabilitation consisted of desensitising and counter-conditioning dogs to the approach of other “stimulus” dogs. Most dogs in the rehabilitation group showed a decline in aggression scores when re-tested after the last treatment (day 11), and differed significantly from the control dogs which showed either an increase or no change in aggression scores (U = 8.5, P < 0.01). Rehabilitation dogs also showed lower frequencies of aggressive body postures (“facing the stimulus dog”, P < 0.05, and “stiff posture”, P < 0.10) and higher frequencies of less assertive postures (“ears back”, P < 0.05, and “lowered neck”, P < 0.10) on day 11. The differences between groups were no longer significant when a reduced sample of dogs was tested 1 week after rehabilitation ended (day 18). The study shows short-term reduction of inter-dog aggression through rehabilitation, but further work is needed on effective ways of maintaining the behavioural change.     

Women's intercollegiate volleyball and tennis: Effects of warm-up, competition, and practice on saliva levels of cortisol and testosterone

In virtually all sports, participants “warm-up” prior to formal competition. Women athletes from a highly ranked varsity college volleyball team and, in a second study, a highly ranked varsity college tennis team gave saliva samples before warm-up, at mid-warm-up (volleyball) or after warm-up (tennis), and immediately after intercollegiate competition. For volleyball and tennis, warm-up was associated with a substantial elevation in saliva levels of testosterone which was carried over through the period of actual competition. Cortisol levels were relatively unchanged during warm-up, but typically rose during competition. Thus, as women prepare for athletic competition by warming up, testosterone levels rise in apparent anticipation of the coming contest and then remain high through the period of play. In volleyball and tennis, after-practice testosterone level was significantly higher than before-practice level, and practice session increases in testosterone (but not cortisol) were positively correlated with increases in testosterone during intercollegiate competition. When practice and competitive play share as yet undetermined key elements, individual differences in this endocrine response to “competition” appear stable across practice and intercollegiate competition.     

Distribution characteristics of salivary cortisol measurements in a healthy young male population

Background

Salivary cortisol has been used in various fields of science as a non-invasive biomarker of stress levels. This study offers the normative reference values of cortisol measurement for healthy young males.

Findings

Salivary cortisol levels were measured in 267 healthy young males (age: 21.7 ± 1.5 years) in the early morning on two consecutive days and were analyzed by radioimmunoassay. Frequency distribution analysis was conducted with mean values of the measurements taken on the 2 days. The mean salivary cortisol level was 20.39 ± 7.74 nmol/l (median: 19.31 nmol/l). The skewness and kurtosis of the distribution of the raw data were 0.72 and 0.68, respectively. They were both improved by a square root transformation but not by a logarithmic transformation.

Conclusions

The skewness of the distribution for salivary cortisol measured in the early morning is considerably smaller than that previously reported from afternoon measurements. A “floor effect” may be an explanation for the difference in the distribution characteristics of salivary cortisol.     

Individual differences in the diurnal cortisol response to stress

The objectives of this study were to explore individual differences associated with diverse reactions in cortisol secretion under different stress levels. This study was part of a larger project concerning working hours and health. Thirty-four white-collar workers participated under two different conditions; one work week with a high stress level (H) and one with a lower stress level (L) as measured through self-rated stress during workdays. Based on the morning cortisol concentration during a workday subjects were divided into two groups. One group consisted of subjects whose morning level of cortisol increased in response to the high-stress week, compared to their morning levels in the low-stress condition (Group 1). The other group consisted of subjects whose morning cortisol response was the opposite, with a lower level under the high stress condition (Group 2). Subjects wore actiwatches, completed a sleep diary, and rated their sleepiness and stress for one work week in each condition, i.e., high and low stress. Saliva samples for measures of cortisol were collected on a Wednesday. Group 2 reported higher workload, fatigue, and exhaustion during both weeks. Since there were no differences in perceived stress, neither within nor between groups, the data indicate that there are other factors influencing morning cortisol. The results suggest that one component modulating the cortisol response might be the level of exhaustion, probably related to work overload. Higher levels of stress in exhausted individuals might suppress morning cortisol levels.     

Blood contamination and the measurement of salivary progesterone and estradiol

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary reproductive hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration and an immunoassay for transferrin. Levels of progesterone increased, and levels of estradiol decreased, in saliva after microinjury. These changes were present only immediately after microinjury. The findings have implications for the use of salivary assays in biobehavioral research, short-term dynamic investigations, pharmacokinetic analyses, and studies of chronobiological changes in progesterone and estradiol levels.     

Effect of dietary fibre type on physical activity and behaviour in kennelled dogs

Dog diets may differ in their effectiveness of maintaining satiety after a meal. Consequently, sensations of hunger, feeding motivation, physical activity, and sensitivity to environmental stressors may be increased. Dietary fibre may be effective in prolonging postprandial satiety depending on type and inclusion level. This study evaluated the effect of fibre fermentability on behaviour in dogs. Sixteen healthy adult dogs were housed individually and fed a low-fermentable fibre (LFF) diet containing 8.5% cellulose or a high-fermentable fibre (HFF) diet containing 8.5% sugar beet pulp and 2% inulin. Dogs were fed two equal portions at 8:30 and 18:30 according to energy requirements. Behaviour of dogs in their home-cage was recorded and analyzed by instantaneous scan sampling (2 × 24 h with 15 min intervals) and focal sampling continuous recordings (10 min per animal per hour, from 9:00 until 18:00). Dogs were subjected to a behaviour test composed of the subtests open-field, sudden-silence, novel-object, and acoustic-startle. The behavioural responses of each dog were recorded. Scores for the scan and focal samples were expressed per clock hour and DIET × TIME effects were tested statistically using Residual Maximum Likelihood (REML). Data from the tests were examined using principal component analysis resulting in the compilation of two components. Data were tested statistically for DIET and DIET × SUBTEST effects using REML. Variables specific for the open-field and novel-object test were analyzed using analysis of variance. For the scans, a significant DIET × TIME effect was found for resting. At night and in the morning, HFF dogs rested more compared to LFF dogs, but they rested less between 14:00 and 17:00. For the continuous recordings, the main findings were a tendency for DIET × TIME effect for time spent resting with a pattern consistent with that for the scans. The interaction was significant for inactive-alert (lie with head up or sitting) with HFF-fed dogs having lower values around 10:00–11:00 and higher values hereafter. Finally, time spent tail wagging was significantly higher for LFF-fed dogs just before the evening meal that may indicate higher level of arousal. For the behaviour tests, no significant DIET or DIET × SUBTEST effects were detected. It is concluded that compared to the LFF diet, the HFF diet increased inactivity in kennelled beagle dogs likely through the prolongation of postprandial satiety. This effect did not change the reaction to stressful events in kennelled laboratory dogs. Enhanced susceptibility to environmental stressors at times of hunger in sensitive companion dogs may occur but requires further study.     

Individual differences in preschoolers' salivary cortisol and alpha-amylase reactivity: Relations to temperament and maladjustment

We examined the relations of 84 preschoolers' (43 boys; mean age = 54 months) situational stress reactivity to their observed emotions and mothers' reports of temperament and adjustment. Salivary cortisol and salivary alpha-amylase (sAA) were collected prior to, and following, a frustrating task. Children's anger, sadness, and positive affect were measured, and mothers reported on preschoolers' dispositional emotionality, regulation, impulsivity, and problem behaviors. Forty-seven percent of children had an increase in sAA and 52% had an increase in cortisol following the challenging task. On average, sAA levels showed the predicted pattern of rise following the frustrating task, followed by return to baseline. For cortisol, there was a mean increase from pre-task to 40 min post-test. sAA reactivity was associated with relatively low levels of dispositional anger and impulsivity and relatively high regulation, particularly for girls. sAA reactivity also was related to low externalizing problems for girls, but not boys. Although cortisol reactivity was unrelated to children's emotions and maladjustment, it was positively related to mothers' reports of regulation. The findings suggest that sAA reactivity in response to a frustrating social task may reflect girls' constrained behavior.     

Shuttlebox avoidance in rhesus monkeys: effects on plasma cortisol and beta-endorphin

Groups of monkeys either extensively pretrained to avoid shocks in a shuttlebox or with minimal prior experience were compared for plasma cortisol and beta-endorphin levels immediately following: (1) an exposure to the box with no shock, (2) the box providing repeated inescapable shocks or (3) a re-exposure to the box, again with no shock presentation. Mere exposure to the unfamiliar box elevated plasma cortisol just as much as exposure + shock did when inexperienced monkeys were tested. However, animals with a history of previously successful shock avoidance showed smaller elevations when exposed to the box alone, than they did when inescapable shock was received. Plasma beta-endorphin levels following shuttlebox exposure showed only a sporadic pattern of elevations in either inexperienced or pretrained monkeys. However, levels of beta-endorphin as determined under control conditions in the home cage were lower in pretrained animals, as were plasma levels of cortisol. The results indicate that behavioral factors may effect plasma cortisol and beta-endorphin following both acute and chronic shuttlebox experience.     

Salivary cortisol changes in humans after winning or losing a dominance contest depend on implicit power motivation

In two studies, one with an all-male German sample and the other with a mixed-sex U.S. sample, subjects competed in pairs on reaction time-based cognitive tasks. Participants were not aware that contest outcome was experimentally varied. In both studies, implicit power motivation, defined as the non-conscious need to dominate or have impact on others, predicted changes in salivary cortisol from before to after the contest. Increased cortisol post-contest was associated with high levels of power motivation among losers but with low levels of power motivation among winners, suggesting that a dominance success is stressful for low-power individuals, whereas a social defeat is stressful for high-power individuals. These results emerged only in participants tested in the afternoon, possibly because of greater variability in cortisol in the morning due to the rapid decline after the morning peak. These studies add to the evidence that individual differences greatly influence whether a social stressor like losing a contest activates the HPA axis in humans.     

Urinary cortisol analysis for monitoring adrenal activity in elephants

Cortisol was measured in dichloromethane-extracted elephant urine using an ¹²⁵I solid-phase radioimmunoassay (RIA). The cortisol RIA was validated by demonstrating 1) parallelism between dilutions of pooled urinary extracts and the standard curve, 2) significant recovery of exogenous cortisol added to elephant urine, and 3) a relationship between changes in peripheral and urinary cortisol after an adrenocorticotropin hormone (ACTH) challenge. One African (Loxodonta africana) and one Asian (Elephas maximus) elephant were given three injections of ACTH (1.25 mg) at 2 h intervals. Serum cortisol increased four- to eightfold within 30 min after the first injection and peaked (nine- to twelvefold increase) after the second injection. Serum concentrations began to decline 2–3 h after the last injection but were still approximately fourfold higher than baseline at the end of the collection period (hour 8). In the urine, cortisol concentrations were increased in the first sample postinjection (1.5–4 h) and peaked twenty- to fortyfold by ～6 h. Urinary cortisol remained elevated at 8 h, but returned to baseline the following morning. Analysis of high performance liquid chromatography fractions of extracted urine revealed that immunoactivity was associated with free cortisol (～90% of total immunoactivity) and a more polar, unidentified metabolite. A method for preserving urine was developed to allow storing unfrozen samples. One pool of urine from each of one African and two Asian elephants was divided into aliquots, placed in tubes containing absolute ethanol (10%), sodium azide (0.1%) or distilled water (control), and frozen after 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 weeks of storage at ～25°C. In unpreserved samples, cortisol concentrations were reduced 46% by 2 weeks and 95% by 24 weeks. In contrast, ethanol- and sodium azide-preserved samples retained 100 and 95% of cortisol immunoactivity through 8 weeks and 93 and 85% of activity through 12 weeks, respectively. We infer from these data that changes in urinary cortisol excretion in the elephant reflect fluctuations in adrenal activity and may be a useful indicator of stress. Additionally, urine samples can be collected and stored unfrozen for at least 2 months before any appreciable loss in cortisol immunoactivity occurs, a finding potentially useful to field application of this technique. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

.     

A Review of Methods for Non-Invasive Heart Rate Measurement on Wrist

When evaluating general health condition on a patient, heart rate is an essential indicator as it is directly representative of the cardiac system state. Continuous measurement methods of heart rate are required for ambulatory monitoring involved in preliminary diagnostic indicators of cardiac diseases or stroke. The growing number of recent developments in wearable devices is reflective of the increasing demand in wrist-worn activity trackers for fitness and health applications. Indeed, the wrist represents a convenient location in terms of form factor and acceptability for patients. While most commercially-available devices are based on optical methods for heart rate measurement, others methods were also developed, based on various physiological phenomena. This review is focused on heart rate measurement methods located on forearm and more specifically on the wrist. For each method, the physiological mechanism involved is described, and the associated transducers for bio-signal acquisition as well as practical developments and prototypes are presented. Methods are discussed on their advantages, limitations and their suitability for an ambulatory use. More specifically, the superposition of motion artefacts over the signal of interest is one of the largest drawbacks for these methods, when used out of laboratory conditions. As such, artefact reduction techniques proposed in the literature are also presented and discussed.     

Distribution and roles of aquaporins in salivary glands

Salivary glands are involved in secretion of saliva, which is known to participate in the protection and hydratation of mucosal structures within the oral cavity, oropharynx and oesophagus, the initiation of digestion, some antimicrobial defence, and the protection from chemical and mechanical stress. Saliva secretion is a watery fluid containing electrolytes and a mixture of proteins and can be stimulated by muscarinic and adrenergic agonists. Since water movement is involved in saliva secretion, the expression, localization and function of aquaporins (AQPs) have been studied in salivary glands. This review will focus on the expression, localization and functional roles of the AQPs identified in salivary glands. The presence of AQP1, AQP5 and AQP8 has been generally accepted by many, while the presence of AQP3, AQP4, AQP6 and AQP7 still remains controversial. Functionally, AQP5 seems to be the only AQP thus far to be clearly playing a major role in the salivary secretion process. Modifications in AQPs expression and/or distribution have been reported in xerostomic conditions.     

The role of salivary cortisol and DHEA-S in response to sexual, humorous, and anxiety-inducing stimuli

Stress and anxiety are commonly thought to be detrimental to sexual function. Several studies in both the human and animal literature, however, have found that inducing anxiety can enhance sexual function in women. The mechanisms that explain a negative relationship between physical and psychological stress and sexual functioning are well documented, but little is known about how stress or anxiety might have a facilitatory effect on sexual arousal. As an initial step in exploring the relationship between anxiety and sexual arousal, the present study examined the role of the autonomic nervous system, and the adrenal hormones cortisol and dehydroepiandrosterone-sulfate (DHEA-S) in response to a sexual film, an anxiety-inducing film, and a humorous film. Nineteen premenopausal women (mean age 24.4 years) who were free from sexual difficulties came into the lab on three separate days. At each session they were shown an anxiety-inducing, sexually arousing, or humorous (control) film while their physiological arousal was measured. They also provided saliva samples before and after each film. Cortisol significantly decreased, while DHEA-S increased in the sexual and humorous conditions. Neither hormone changed significantly in the anxiety-inducing condition. Autonomic nervous system activity measured by heart rate and heart rate variability did not change in response to the sexual or anxiety-inducing films, but heart rate variability increased significantly in response to the humorous film. The cortisol/DHEA-S ratio at the post-sexual film time point was significantly negatively correlated with genital arousal (measured by vaginal pulse amplitude). Anxiety-inducing films did not result in a physiological stress response, which can explain why they do not impair sexual function.     

Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement.

Cortisol 3-(o-carboxymethyl)oxime (C3-CMO) and a commercially available biotin-hydrazide derivative were used to synthesize a C3-CMO-biotin conjugate. C3-CMO was converted into a N-hydroxysuccinimide ester derivative which in a second reaction step was allowed to interact with the hydrazide derivative of biotin. This simple-to-perform synthesis yielded a conjugate suitable for use as a tracer in immunoassays for cortisol measurement. Employing biotin as the primary probe in a competitive solid phase immunoassay allows for variable end point determination by means of commercially available labeled avidin or streptavidin derivatives. Streptavidin-Europium was used in conjunction with the DELFIA-system for time-resolved fluorometric end point measurement (TR-FIA) throughout the study. In addition, colorimetric end point determination (ELISA) using streptavidin-alkaline phosphatase as a secondary probe was established and evaluated. Both forms of this non-isotopic assay showed excellent correlation with a commercially available radioimmunoassay adapted for salivary cortisol measurement. The lower detection limit was 0.43 nM for a 50 microliters salivary sample. The intra-assay coefficient of variation was 6.7, 4.7 and 4.0% at cortisol concentrations of 2.2, 5.5 and 13.2 nM, respectively (n = 37), and the corresponding inter-assay coefficients of variation were 9.0, 8.6 and 7.1% (n = 50). The competitive immunoassay requires 1.5 h incubation time and shows robust and reproducible performance. The C3-CMO-biotin conjugate allows for sensitive and flexible end point determination of salivary cortisol levels in immunoassays.