Habituation Trials Research Articles

The reliability of the IL-6, sIL-6R and sgp130 response to a preloaded time trial

Interleukin-6 (IL-6) is a cytokine that can mediate numerous biological actions including fatigue. Circulating IL-6 increases during prolonged exercise, and furthermore, the signalling receptors sIL-6R and sgp130 are also increased. The variability of the response of these markers to exercise is unknown; therefore, we examined the changes in these markers to a preloaded time trial bout of running. Nine males performed three identical trials where participants ran at 60% vVO2max for 2 h interspersed with 30 s at 90% vVO2max every 10 min, followed by a 5-km time trial. Blood samples were drawn at baseline, following the 2-h bout, post time trial, 1 h post time trial and the following morning. Results showed that between-subject variability (CVg) was greater than within-subject variation (CVi) for the three markers. IL-6, sIL-6R and sgp130 demonstrated a CVi of 15.3-25.5%, 15.0-17.6% and 6.2-9.4% variation, respectively, across the time points. When the data from the second and third trials were analysed independently, CVi was reduced which is supported by the time trial results for which CVi improve (4.7-2.4%). In conclusion, the results indicate that a large variation in response to exercise can be reduced following a habituation trial.

Lesion of infralimbic cortex occludes stress effects on retrieval of extinction but not fear conditioning

Chronic stress produces dendritic retraction in medial prefrontal cortex and impairs retrieval of extinction of conditioned fear, a behavior mediated by the infralimbic region (IL) of medial prefrontal cortex. To test the hypothesis that stress-induced changes in IL contribute to the stress-induced impairment in extinction retrieval, we performed an occlusion experiment in which we assessed the effects of stress alone, lesion of IL alone, and the combined effects of stress and lesion on extinction retrieval. If IL is the substrate upon which stress acts to produce deficits in extinction retrieval, then prior removal of IL should prevent the effect of stress on extinction retrieval. Rats received either sham or ibotenic acid lesions of IL. Rats in each group then remained unstressed or underwent daily restraint stress for 1 week. Following the final day of restraint, rats received five habituation trials to a 30-s tone, followed by seven pairings of the tone with a 500-ms coterminating footshock. One hour later, rats received tone-alone extinction trials. On the following day, rats were given two extinction trials to test for extinction retrieval. Percent freezing was assessed throughout. Stress increased freezing during conditioning, and IL lesion did not block this effect. Either IL lesion alone or stress alone increased freezing on initial extinction trials. IL lesion did not attenuate the effect of stress during initial extinction. Similarly, IL lesion alone and stress alone produced deficits in extinction retrieval. However, stressed rats with IL lesions showed extinction retrieval comparable to that seen in unstressed, sham-lesioned rats. Thus, lesion of IL occluded the stress-induced impairment of extinction retrieval but failed to prevent the stress-induced facilitation of fear conditioning. This dissociation suggests that the effects of stress on these two aspects of emotion regulation are mediated at least in part by independent mechanisms, and that stress-induced changes in IL contribute to stress-induced deficits in extinction retrieval.

Long‐term recognition memory of individual conspecifics is associated with telencephalic expression of Egr‐1 in the electric fish Apteronotus leptorhynchus

Primates and songbirds can learn to recognize individual conspecifics based on complex sensory cues; this requires a large, highly differentiated dorsal telencephalon. Here we show that the electric fish Apteronotus leptorhynchus can learn to recognize individual conspecifics based on a simple cue, the beat frequency of their summed sinusoidal electric organ discharges (EOD). Male fish produce transient communication signals (chirps) in response to mimic EODs. The chirp response habituates over repeated stimulus presentations within one experimental session, continues to habituate over successive daily sessions and is nearly extinguished after 5-7 days. Habituation of the chirp response was specific to the presented beat frequency. The conversion of short- to long-term habituation could be disrupted by cooling the head 30 minutes after the daily habituation trials. Consolidation of long-term memory in mammals is thought to involve induced expression of an immediate early gene, Egr-1. We cloned the Apteronotid homolog of the Egr-1 gene and found that chirp-evoking stimuli induced strong expression of its mRNA within the dorsal (Dd), central (DC), and lateral (DL) subdivisions of the dorsal telencephalon. Interestingly, the dorsolateral region is hypothesized to be homologous to the amniote hippocampal formation. We conclude that A. leptorhynchus can learn to identify individual conspecifics based on their EOD frequency and can remember these frequencies for several days. We hypothesize that this form of learning, as in primates and songbirds, requires a subset of dorsal telencephalic areas and involves a consolidation-like process that includes the expression of the transcription factor AptEgr-1.

Influences of information processing and disengagement in infants' looking behaviour

AbstractThe present study considers the joint influences of information processing and disengagement in looking behaviour within a habituation paradigm. Six‐month‐old infants were habituated, during which their heart rate (HR) was measured. A parametric model of habituation yielded for each infant parameter estimates of their habituation performance. These parameters were interpreted as assessing information processing and disengagement. Corresponding measures were obtained from the HR data. The HR measures and habituation model parameter estimates were significantly correlated, as predicted. In addition, an attention getter, presented prior to each habituation trial, influenced indicators of information processing, but not of disengagement. Results confirmed the advantages of a modelling approach. In addition, and more importantly, findings led to the conclusion that both information processing as well as disengagement are involved in infants' looking behaviour in visual habituation. Copyright © 2009 John Wiley & Sons, Ltd.

Effects of starting strategy on 5-min cycling time-trial performance

The importance of pacing for middle-distance performance is well recognized, yet previous research has produced equivocal results. Twenty-six trained male cyclists ([Vdot]O2peak 62.8 ± 5.9 ml · kg−1 · min−1; maximal aerobic power output 340 ± 43 W; mean ± s) performed three cycling time-trials where the total external work (102.7 ± 13.7 kJ) for each trial was identical to the best of two 5-min habituation trials. Markers of aerobic and anaerobic metabolism were assessed in 12 participants. Power output during the first quarter of the time-trials was fixed to control external mechanical work done (25.7 ± 3.4 kJ) and induce fast-, even-, and slow-starting strategies (60, 75, and 90 s, respectively). Finishing times for the fast-start time-trial (4:53 ± 0:11 min:s) were shorter than for the even-start (5:04 ± 0:11 min:s; 95% CI = 5 to 18 s, effect size = 0.65, P < 0.001) and slow-start time-trial (5:09 ± 0:11 min:s; 95% CI = 7 to 24 s, effect size = 1.00, P < 0.001). Mean [Vdot]O2 during the fast-start trials (4.31 ± 0.51 litres · min−1) was 0.18 ± 0.19 litres · min−1 (95% CI = 0.07 to 0.30 litres · min−1, effect size = 0.94, P = 0.003) higher than the even- and 0.18 ± 0.20 litres · min−1 (95% CI = 0.5 to 0.30 litres · min−1, effect size = 0.86, P = 0.007) higher than the slow-start time-trial. Oxygen deficit was greatest during the first quarter of the fast-start trial but was lower than the even- and slow-start trials during the second quarter of the trial. Blood lactate and pH were similar between the three trials. In conclusion, performance during a 5-min cycling time-trial was improved with the adoption of a fast- rather than an even- or slow-starting strategy.

Humans and natural predators induce different fear/anxiety reactions and response pattern to diazepam in marmoset monkeys

The behavioral response of marmoset monkeys in the Human Threat (HT) test of anxiety, and the effects of diazepam (DZP), were compared to those in the Predator Confrontation (PC) procedure. Subjects ( n = 13) were initially submitted to four habituation trials, followed by four random confrontation sessions for each test (DZP 0, 1, 2 and 3 mg/kg). Each trial was divided into three consecutive 5-min intervals: pre-exposure, exposure (human observer, taxidermized oncilla cat) and post-exposure. As DZP induced sedation, marmosets ( n = 10) were re-tested in a second experiment, consisting of two habituation trials and four confrontation sessions per stimulus, with lower DZP doses (0, 0.10, 0.25 and 0.50 mg/kg). Exposure to both stimuli significantly increased direct gazes and alarm calls, being dose-dependently reduced by DZP only in the PC test. In the HT protocol, the significant decrease in aerial scans was not detected with 0.10 mg/kg DZP. Locomotion, proximity, displacement activities and vigilance were not consistently influenced by the stimuli and/or DZP. The results thus suggest that the HT test had a greater impact on the marmosets' behavior, while DZP was more effective on the reactions observed in the PC test, possibly due to the inherent nature of each stimulus, distinct threat levels and/or presentation order.

Variability Of Plasma Interleukin-6, Soluble Interleukin-6 Receptor And Soluble Glycoprotein 130 Response To A Run Pre-load Time Trial Protocol

During exercise, IL-6 is actively produced. Elevations in IL-6 levels can impair athletic performance and increase sensation of fatigue. Nutritional interventions can alter the IL-6 response to exercise but the impact this may have on performance is yet to be investigated. To test the hypothesis that an altered IL-6 response to exercise influences athletic performance, studies should employ an exercise preload followed by a time trial. PURPOSE: The aim of this study was to provide data on the variability of IL-6 and its signalling molecules; sIL-6R and sgp130 response to a specific pre-load time trial protocol. METHODS: Ten trained fasted runners with mean (SD) age: 27 (5) yr, body mass: 72.4 (8) kg, VO2max 59 (5) ml-1kg-1min. completed a pre-load of 120 min running at 60% VO2max and followed by a 5 km time trial on 3 occasions separated by one week. The first trial was a habituation trial. Venous blood samples were drawn prior to exercise, immediately following both the pre-load and time trial. Plasma samples were analysed in duplicate for IL-6, sIL-6R and sgp130. RESULTS: Coefficient of variation between trial A and B for time to complete 5 km was 3.8% (see Table 1 for C of V% for IL-6, sIL-6R and sgp130).Table 1: Coefficient of variation % in plasma parameters between trialsCONCLUSION: Despite attempts to standardise conditions such as pre-exercise glycogen levels, food intake and exercise training between trials, our results indicate that there is considerable variability in the IL-6, sIL-6R, sgp130 response to exercise. This variability must be taken into account in future studies attempting to explore the relationship between these variables and performance using a pre-load time trial protocol. Supported by Biotechnology and Biological Research Council (UK) and GlaxoSmithKline Nutritional Healthcare (UK).

Infant perception of static two-dimensional transparency information

The present habituation – dishabituation study focused on infants' perception of static two-dimensional transparency displays. Infants 4 and 8 months of age were habituated to a transparency display. In this stimulus, each corner of a semi-transparent square surface covered a quadrant of a circle. During the posthabituation period, the infants were presented with the semi-transparent square overlay from the habituation display and a (non-transparent) square area cut out of the habituation stimulus. The 8-month-olds looked significantly longer at the non-transparent surface, meaning that they apparently recognized the other test pattern as the semi-transparent filter they had also seen during the habituation trials, and that they regarded the non-transparent test target as being novel. In contrast, the younger participants did not exhibit a novelty preference during the posthabituation trials. Control conditions tested the impact of spontaneous stimulus preferences on the results. The findings are discussed within the framework of infant pictorial depth perception. They provide further evidence for the hypothesis that infants' sensitivity to pictorial depth cues emerges between 4 – 5 and 7 – 8 months of life.

Infants' ability to parse continuous actions.

In a series of 3 experiments, the authors examined 6- and 8-month-old infants' capacities to detect target actions in a continuous action sequence. In Experiment 1, infants were habituated to 2 different target actions and subsequently were presented with 2 continuous action sequences that either included or did not include the familiar target actions. Infants looked significantly longer at the sequences that were novel. Experiment 2 presented the habituation and test trials in the reverse order. The results showed that infants habituated to the sequence still showed reliable evidence of recognizing the target action during the test trials. Experiment 3 was comparable to Experiment 2, except it tested whether infants could detect a different event segment, namely the transitions between events. The results showed that infants did not discriminate between test trials suggesting that transitions between events are not as easy for infants to recognize.

Brief neonatal maternal separation alters extinction of conditioned fear and corticolimbic glucocorticoid and NMDA receptor expression in adult rats

Neonatal maternal separation alters adult HPA axis responsiveness to stress, adult emotionality, and glucocorticoid receptor (GR) concentrations in forebrain regions such as hippocampus. To investigate effects of neonatal maternal separation on emotion regulation and its neural substrates, we assessed acquisition and extinction of conditioned fear in adult rats that underwent neonatal maternal separation. Corticolimbic structures including basolateral amygdala and medial prefrontal cortex are critical for acquisition and extinction of conditioned fear, and such learning is N-methyl-D-aspartic acid (NMDA) receptor-dependent. Thus, we used immunohistochemistry to assess expression of the GR and the NR1 subunit of the NMDA receptor in basolateral amygdala and medial prefrontal cortex. On postnatal days 2-14, pups underwent control rearing or maternal separation for 15 min per day. Fear conditioning and extinction in adulthood were then assessed in male rats. Rats received five tone-alone habituation trials, then seven tone/footshock pairings. After 1 h, rats received tone-alone extinction trials to criterion, and 15 recall of extinction trials the next day. Brains were processed for immunohistochemical labeling of GR and NR1, and staining was quantified. Brief maternal separation did not alter acquisition or initial extinction, but impaired extinction recall. Brief maternal separation did not alter GR or NR1 expression in basolateral amygdala. However, brief maternal separation increased GR and decreased NR1 expression specifically in the infralimbic region of medial prefrontal cortex, consistent with work implicating this area in extinction recall. Thus, brief maternal separation impaired extinction recall and altered GR and NR1 expression in its neural substrate in adults.

Properties of perception of laterally moving objects in persons with profound retardation.

Twenty-two persons with profound retardation were assessed for their perception of moving objects at a velocity of 5 degrees s-1. Abnormalities were not found in the ocular structures and optic discs of subjects upon ophthalmological examination. Stimuli consisted of six habituation trials and two test trials. Duration of eye movement in pursuit of stimulus was estimated by calculating the linearity and gradients of electro-oculograms. Subjects were divided into three groups based on the change of estimated time of pursuit eye movement. Those showing habituation as well as dishabituation (group 1) had higher developmental communicative ages than subjects with only habituation (group 2) and subjects who did not exhibit habituation (group 3). The results indicated a relationship between the perception of moving objects and the development of communication level in persons with profound retardation. The present study investigated abnormalities in the computer tomographic scanning of the subjects' heads and found cerebral disturbance of the visual cognition of moving objects in persons with profound retardation.

Changes in experimental conditions alter anti-predator vigilance and sequence predictability in captive marmosets

Anti-predator vigilance and its sequence predictability in captive adult male black tufted-ear marmosets (Callithrix penicillata) was analyzed under three experimental conditions: (1) four novel (maze) environment habituation trials; (2) six taxidermized (cat) predator confrontation trials in the same maze environment; and (3) four additional maze-only trials, in the absence of the cat stimulus. Significant quantitative and qualitative differences (aerial vs. terrestrial) were observed in scan and glance behaviors within and between the three experimental conditions. Furthermore, inter-scan bout sequence significantly deviated from randomness during the initial maze habituation and predator confrontation trials, as well as during all predator removal trials. This parameter, however, followed a random pattern during the course of the remaining sessions. Therefore, vigilance in marmosets seems to be an important and highly organized component of this species' anti-predation repertoire, inasmuch as it occurs at high rates, alters according to specific environmental cues and has a subtle differential adaptive response after repeated trials.

Discrimination of Individuals by Odor in Male Mongolian Gerbils, Meriones unguiculatus

The ability to discriminate among individuals plays a fundamental role in the establishment of social relationships in animals. We examined how Mongolian gerbils (Meriones unguiculatus) discriminate among individuals using odor. In the first experiment, the ability of male gerbils to discriminate among five odor sources from different individuals was investigated using a habituation-dishabituation paradigm. After male gerbils had been habituated to a scent from one individual, they were exposed to familiar and unfamiliar scents from different donors simultaneously. Where urine and ventral gland secretions were used, the subjects spent more time investigating novel odors than familiar ones, suggesting that they were able to discriminate individual differences in these odor sources. However, with the scents of feces and saliva, they could detect, but could not discriminate individual differences; with scent from inside the pinnae, they could not even detect. In the second experiment, we tested whether cross-habituation occurred between the scents of urine and ventral gland secretions. A male was exposed repeatedly to urine from one of two familiar donor males during four habituation trials, and was then exposed to the ventral gland secretions from two donors simultaneously. The subject males spent more time investigating scents of ventral gland secretions, but there was no difference in the investigation time between ventral gland scents from the two donors. These results suggest that male gerbils discriminate among individuals using odors from urine and ventral gland secretions and that cross-habituation may not occur between these scents during social-memory formation.

Brain Mechanisms Implicated in the Preattentive Categorization of Speech Sounds Revealed Using fMRI and a Short-Interval Habituation Trial Paradigm

A hallmark of categorical perception is better discrimination of stimulus tokens from 2 different categories compared with token pairs that are equally dissimilar but drawn from the same category. This effect is well studied in speech perception and represents an important characteristic of how the phonetic form of speech is processed. We investigated the brain mechanisms of categorical perception of stop consonants using functional magnetic resonance imaging and a passive short-interval habituation trial design (Zevin and McCandliss 2005). The paradigm takes advantage of neural adaptation effects to identify specific regions sensitive to an oddball stimulus presented in the context of a repeated item. These effects were compared for changes in stimulus characteristics that result in either a between-category (phonetic and acoustic) or a within-category (acoustic only) stimulus shift. Significantly greater activation for between-category than within-category stimuli was observed in left superior sulcus and middle temporal gyrus as well as in inferior parietal cortex. In contrast, only a subcortical region specifically responded to within-category changes. The data suggest that these habituation effects are due to the unattended detection of a phonetic stimulus feature.

Intrahippocampal injections of Tat: Effects on prepulse inhibition of the auditory startle response in adult male rats

The presence of human immunodeficiency virus (HIV-1) in the brain mediates the pathogenesis of HIV-associated dementia complex (HAD), partially through the viral toxins gp120 and Tat. This study characterized potential deficits in sensorimotor gating, as measured by prepulse inhibition (PPI), following hippocampal administration of Tat. Adult, male Sprague–Dawley rats were bilaterally injected with 50 μg Tat or saline (1 μl volume), into the hippocampus. Following 7 weeks of recovery, all animals were tested using the auditory startle response (ASR) with habituation, control, and PPI trials. Assessment of ASR habituation [100dB(A) white noise stimulus, 70dB(A) background, 5-min acclimation period, 36 habituation trials with fixed interstimulus interval (ISI) of 10 s] demonstrated a significant ∼ 50% reduction in the overall peak ASR amplitude, but no change in peak ASR latency, nor an effect on the rate of habituation. PPI measures demonstrated robust alterations in sensorimotor gating. The PPI test (ISI of 0, 8, 40, 80, 120, or 4000 ms, 6-trial blocks, Latin-square) showed an attenuated response on peak ASR amplitude during the control trials (0 and 4000 ms ISI), but not on the PPI trials (8–120 ms ISI). Most striking was the rightward shift in ISI for maximal inhibition of the response ( χ 2(1) = 4.7, p ≤ 0.03). There was no significant peak ASR latency effect during the control trials (< 1 ms) of the PPI test, although there was the suggestion of a slowing of the response (4 ms, ∼ 15%) across PPI trials. Collectively, the present data suggest that intrahippocampal injections of Tat have adverse effects on cognitive processing, as indexed by sensorimotor gating.

Individual Odour Similarity and Discrimination in the Coruro (Spalacopus cyanus, Octodontidae)

AbstractPrevious studies of subterranean, fossorial and above‐ground rodents have demonstrated that similarities in individual odours covary with genetic similarities thus supporting the theory of odour‐genes covariance (i.e. the closer the individuals are genetically, the greater the similarities between their odours). We used the habituation‐generalization paradigm, where the subject is exposed to the same odour stimulus in three consecutive habituation trials followed by two test trials in which the odour from two other individuals are presented successively. Using this test design, we showed that the socially living coruros (Spalacopus cyanus) discriminate individuals on the basis of their ano‐genital odours and also respond to odour similarities between individuals. Male and female subjects treated ano‐genital odours of two same‐sex family members and also the odours of two sibling strangers as different to each other. At the same time, they treated the odours of siblings as similar compared with the odour of an unrelated individual. No gender differences were detected. Our results contrast with those from other rodent species that did not spontaneously discriminate between individual odours of siblings from a different family than their own. The polygyneous lifestyle may provide the selective background for that difference. Additional research will be necessary to explore this hypothesis and to rule out differences due to dietary preferences and due to the type of paradigm chosen for the tests.

Effectiveness of a Motion-Activated Laser Hazing System for Repelling Captive Canada Geese

Abstract Effective management techniques are needed to disperse Canada geese (Branta canadensis) and reduce the human–wildlife conflicts associated with high population densities. We evaluated the effectiveness of a motion-activated laser hazing system for repelling captive Canada geese. The system decreased occupancy of 8 pairs of geese on the treated subplot by 83% during habituation trials. When an additional pair of geese were added to the experiment, occupancy of the treated subplot decreased >92% during each of the 20 nights of the extended habituation test. Avoidance (conditioned during the test) remained <80% of pretreatment levels during the 2 days immediately following the habituation test but extinguished 3 days subsequent to the permanent inactivation of the laser hazing system. The motion-activated laser hazing system effectively repelled Canada geese in captivity. Additional field research is needed to determine the spatial extent of the laser hazing system and the effectiveness of the Doppl...

Using dynamic field theory to rethink infant habituation.

Much of what psychologists know about infant perception and cognition is based on habituation, but the process itself is still poorly understood. Here the authors offer a dynamic field model of infant visual habituation, which simulates the known features of habituation, including familiarity and novelty effects, stimulus intensity effects, and age and individual differences. The model is based on a general class of dynamic (time-based) models that integrate environmental input in varying metric dimensions to reach a single decision. Here the authors provide simulated visual input of varying strengths, distances, and durations to 2 coupled and interacting fields. The 1st represents the activation that drives "looking," and the 2nd, the inhibition that leads to "looking away," or habituation. By varying the parameters of the field, the authors simulate the time course of habituation trials and show how these dynamics can lead to different depths of habituation, which then determine how the system dishabituates. The authors use the model to simulate a set of influential experiments by R. Baillargeon (1986, 1987a, 1987b) using the well-known "drawbridge" paradigm. The dynamic field model provides a coherent explanation without invoking infant object knowledge. The authors show that small changes in model parameters can lead to qualitatively different outcomes. Because in typical infant cognition experiments, critical parameters are unknown, effects attributed to conceptual knowledge may be explained by the dynamics of habituation.

Chronic stress impairs recall of extinction of conditioned fear

Chronic restraint stress produces retraction of apical dendrites of pyramidal neurons in medial prefrontal cortex. To begin to examine the functional significance of this dendritic reorganization, we assessed the effects of chronic restraint stress on a prefrontally mediated behavior, extinction of conditioned fear. After bar press training to obtain a baseline of activity against which to measure freezing, rats were either unstressed or stressed via placement in a plastic restrainer (3 h/day for 1 week). After an additional day of bar press training, rats underwent fear conditioning and extinction. Rats received five habituation trials to a 30-s tone (4.5 kHz, 80 db) followed by seven pairings of tone and footshock (500 ms, 0.5 mA). One hour later, rats received tone-alone extinction trials to criterion. The next day, rats received 15 additional extinction trials. Percent freezing was assessed during all phases of training. Stress did not significantly affect unconditioned responding to tone, acquisition of conditioned fear, or initial extinction, but significantly increased freezing on extinction day 2. Thus, consistent with the regressive dendritic changes seen in medial prefrontal cortex, one week of restraint stress specifically impaired recall of extinction, a pattern of deficits typical of animals with impaired medial prefrontal function.

The reproducibility of an endurance performance test in adolescent cyclists

The purpose of the study was to measure the reproducibility of a performance test in well-trained adolescent cyclists. Eight male and one female cyclist [mean age 15.7 (0.7) y] participated in the study. Lactate threshold (LT) and peak VO2 were assessed. The performance test was repeated on three separate days and consisted of 30 min of steady state (SS) cycling at 80% of individual LT. Immediately after the SS cycling a time trial (TT) started with the cyclists having to complete a fixed amount of work as fast as possible. Reliability was assessed for the TT with the coefficient of variation (CV) as the (SD/mean)*100 for each participant, intraclass correlation coefficients (ICC) and typical error (SD of the difference in mean /radical2). The group mean (SD) times for the TT were TT1 1889 (306), TT2 1857 (283) and TT3 1953 (279) s respectively. Individual CV varied from 0.25% to 10%. The ICC for TT1/2 and 2/3 were r = 0.78 and 0.93 (P<0.05). The typical errors, expressed as a CV% on the log transformed performance times, were 7.3 and 3.7% for TT1/2 and TT2/3 respectively. The largest individual CVs were observed between TT1 and TT2. The differences in CV and SD among the three TTs indicate that trial two and three were more reliable than TT1, suggesting a habituation trial is needed. It is concluded that the present performance test is reliable in adolescent cyclists with lower variation between trials 2 and 3.