Participants and Study Design: This was a cross-sectional study whereby males and females between 4 to 6 years old (n = 59) were recruited from Eastern Illinois. Flyers using age appropriate language were placed in local schools, community centers, and summer camps, as well as online social media groups, and were used to recruit participants. Children were screened prior to participation and were excluded based on several factors, including the presence of attentional and developmental disorders (Attention-Deficit/Hyperactivity Disorder, Autism Spectrum Disorder, Down’s Syndrome), uncorrected vision, and hearing loss. To determine if these criteria were met, parents reported if a physician had diagnosed their child with any of the aforementioned factors. There were 61 children screened, and all were eligible. All participants provided verbal and/or written assent, and their guardians provided written consent before enrollment in the study. All study procedures were approved by the University of Illinois at Urbana Champaign Institutional Review Board (IRB #16484) and conformed to the guidelines of the Declaration of Helsinki. Study procedures took place over three laboratory visits. During visit 1, 61 participants completed informed assent forms and underwent anthropometric and adiposity assessments. Participants completed the self-paced 6MWT. Additionally, parents/guardians completed informed consent and surveys to report the child’s health history as well as family and child demographic information (i.e., age, sex, level of education, household income). Parents were also given 7-day diet records to complete on behalf of the child completing the study and were asked to return them at the second visit. During visit 2, 60 participants completed the pencil and paper-based neuropsychological battery (Woodcock Johnson Early Cognitive and Academic Development Test (ECAD™)) for assessment of general intellectual abilities. A subsample of participants (n = 33) completed a selective attention auditory cognitive task while wearing an electrode cap. This subsample was self-selected in that it consisted of children who tolerated the administration of the EEG cap and completed the task with minimal disruptions to the electrodes. During visit 3, 58 participants completed the modified Eriksen flanker task to assess attentional inhibition. The hearts and flowers task was initially administered during visit 3 (62% of participants); however, due to time constraints on visit 3, the hearts and flowers task was moved to visit 1 (38%) and administered approximately 30 min after the 6MWT.
Although the 6MWT was completed by all 59 children who were enrolled in the study, data may be missing due to participants choosing not to complete some tasks or procedures on the day of testing. Procedures for ECAD™ were completed among 53 participants. Furthermore, 48 and 53 children completed the flanker and the hearts and flowers task, respectively. Diet records were only returned by 55 participants and 53 completed the dual-energy X-ray absorptiometry (DXA) procedure. Lastly, 32 participants completed the auditory oddball task with the electrode cap, whereas usable ERP data was available for 26 participants. A few parents chose not to reveal some demographic information, therefore 51 and 52 participants answered questions regarding race and household income, respectively.
Weight Status and Adiposity Assessment: Participants’ height and weight were measured, without shoes, using a stadiometer (model 240; Seca, Hamburg, Germany) and a Tanita WB-300 Plus digital scale (Tanita, Tokyo, Japan), respectively. Each measurement was taken three times, and the average was used for analyses. BMI-for-age-percentile cut-offs from the Centers for Disease Control (CDC) were used to determine weight status for descriptive purposes [44
]. Adiposity was assessed by dual-energy X-ray absorptiometry (DXA) using a Hologic Horizon W bone densitometer (software version 13.4.2, Bedford, MA, USA). VAT was estimated using the standard software measure. This estimated VAT has been shown to correlate (r
= 0.92; p
< 0.01) with computed tomography (CT)-determined VAT values [45
Diet Quality Assessment: Parents completed a 7-day diet record on behalf of their child. Their diet information was analyzed using the Nutrition Data System for Research software (NDSR; Nutrition Coordinating Center, Minneapolis, MN, USA). Healthy Eating Index (Total HEI-2015) scores were derived to determine diet quality. This method has been used in the past for children of the same age group [46
Six-Minute Walk Test (6MWT): The 6MWT was conducted indoors along a flat and straight walkway using guidelines and procedures previously described [38
]. Participants were instructed to wear comfortable shoes that allowed them to be physically active. The course length was 20 m, and cones were placed at either end of the walking course to indicate starting and endpoints. The same instructions were given to all children before undertaking the walk test. Participants were tested individually and were provided with standard instructions that the purpose of the test was to find out how far they could walk in 6 min during the allotted time. Instructions for the task included walking back and forth as fast as they could without running from one cone to another and walking around each cone when reaching it. Standard phrases such as “keep going”, “you are doing really well” as well as announcements of time remaining were provided to participants. No comments were made regarding the child’s performance, such as, ‘‘you could go faster’’ or ‘‘slow down’’ although children were reminded of task instructions when needed (e.g., “remember not to run.”). The total distance (m) was recorded and utilized as the variable of interest in the analyses. A portable pulse oximeter (Nonin, Aeromedix, Jackson Hole, WY, USA) was used to assess each child’s heart rate (HR) and oxygen saturation (SpO2
) at the index finger immediately prior to and following the 6MWT.
Woodcock Johnson Early Cognitive and Academic Development: The ECAD™ is a standardized measure of early academic skills, comprised of 10 tests: seven measuring cognitive abilities and three measuring academic achievements. The ECAD™ produces a comprehensive score, General Intellectual Ability (GIA), as well as two composite scores: Early Academic Skills and Expressive Language. All testing and scoring material was provided by the publisher, including flipbooks, audio recordings, scoring software, and examiner’s manual. This test has been normed and shown to be valid and consistent with other known standardized neuropsychological tests among children [48
]. The test was administered according to standardization procedures by a trained member of the research team.
Hearts and Flowers Task: A hearts and flowers task, previously shown to be appropriate for children as young as 4 years, was used to assess cognitive flexibility [11
]. In all conditions, a white heart or a flower appeared on the right or left of a fixation cross presented on a black computer screen. Participants were asked to indicate the side of the screen either consistent with or in opposition to the stimulus presentation by making a keyboard button press. A different rule set was applied to each stimulus type, such that participants were instructed to indicate the same side of the stimulus appearance for heart stimuli and the opposite side of the stimulus appearance for flower stimuli. The task consisted of three experimental blocks. Participants were given a practice block, where they had to get at least a 70% overall accuracy, to ensure they understand instructions. The first two blocks were considered homogeneous blocks, in which participants were exposed to only one stimulus type (hearts in Block 1 and flowers in Block 2), and thus relied upon only one ruleset. In the third, heterogeneous block, stimuli were mixed, so that participants had to continually switch rule sets eliciting cognitive flexibility, as well as components of working memory and inhibition (Figure 1
). Participants completed two homogeneous blocks of 20 trials each (one showing heart stimuli, the other showing flower stimuli) and one heterogeneous block of 40 trials in which the heart and flower trials were mixed. Across blocks, stimuli were presented for 2150 ms with inter-trial intervals (ITIs) of 1800 or 2200 ms. Outcome measurements for this task included accuracy and reaction time for both the homogeneous and heterogeneous trials.
Eriksen Flanker Task: A modified version of the Eriksen flanker task was used to assess attentional inhibition [49
]. Various versions of the flanker task have proven to be reliable and valid tools to use in preschool-aged children [50
], and have been previously employed in children as young as 4 years as well as school-aged children [51
]. The flanker task requires children to respond to a centrally presented target stimulus amid an array of 4 flanking stimuli. In the version of the task used in the current study, both the target and flanking stimuli were left- or right-oriented fish, illustrated in Figure 2
. The task consisted of congruent trials, in which flanking fish were facing the same direction as the target fish, and incongruent trials, in which flanking fish were facing the opposite direction of the target fish. Participants were given a practice block, where they had to get at least a 70% overall accuracy, to ensure they understood instructions. Successful performance on the incongruent trials, relative to congruent trials, requires cognitive control to selectively attend and respond to the directionality of the target stimulus while suppressing interferences elicited by the flanking stimuli. Congruent and incongruent trials were presented in random order with equal probability based on the congruency and directionality of the target stimulus. Participants completed one block of 50 trials. Stimulus presentation time was 2700 ms with a jittered ITI of 2500, 2700, or 2900 ms. Outcome measures for this task included accuracy and reaction time for the congruent and incongruent trials.
Auditory Oddball Task: A subsample of participants also completed an auditory oddball (AOB) task designed to assess selective attention. The computerized task consisted of 40 high-pitched target tones (1000 Hz) randomly interspersed with 200 low-pitched standard tones (500 Hz). Children were asked to sit as still as possible while wearing an electrode cap and holding a response button box. They were then instructed to hit a button on the box as quickly as possible when they heard the high tone. Tones were played through a speaker system at an approximate decibel level of 60 dB. Participants were given a practice block, where they had to get at least a 70%, to ensure they understood instructions. Outcome measures for this task included accuracy, reaction time, inverse efficiency, and coefficient of variation. Only trials that were correctly responded to were utilized for the behavioral reaction time data. However, ERP data extraction was not behaviorally dependent, and waveforms were extracted for all trials.
ERP Recording: During the AOB task, participants wore a 64 channel Neuro-scan Quikcap to collect encephalographic recording (Compumedics, Charleston, NC, USA). The electrode sites complied with the standard, international 10-10 system. Online, inter-electrode impedances were kept at <10 KΩ; a reference electrode placed between Cz and CPz was used; and the AFz was used as the ground. Continuous data were digitized at a sampling rate of 500 Hz, then amplified 500 times with a direct current to 70-Hz filter, and a 60-Hz notch filter was employed. A Neuroscan Synamps2 amplifier was used. Offline, data were re-referenced to the average of the two mastoid electrodes. A Gratton correction was employed on the midline electrodes to account for noise due to eye blinks. Stimulus-locked epochs were created from −200 to 1500 ms. The −200 to 0 window was used for baseline correction and epochs were filtered using a zero-phase shift low pass filter at 30-Hz. An artifact detection threshold of ±125 μV was employed.
Because ERPs are seldom reported in children within this age range, data were visually inspected to determine time windows and electrodes of interest. To do so, the collapsed localizer method was utilized, in which data from all participants and all conditions were collapsed into a single waveform, which was used for visual inspection [52
]. A negativity resembling the prototypical N2 was observed in the Fz electrode within the time frame of 150–350 ms. A later negativity similar to the prototypical P3 was observed in the Cz electrode from 700–1100 ms. The N2 and P3 waveforms are illustrated in Figure 3
. Thus, while the terms “N2” and “P3” will be used to reference these components throughout this report, it should be noted that the time windows for these components are later than what is typically seen in older children and adults [53
]. The mean amplitude and local peak latency during the time windows specified above were used as metrics of the respective ERP components.
Statistical Analyses: Given that previous research examining the relationship between 6MWT and cognitive function in preschool-aged children is limited [54
], a power calculation (G-Power 22.214.171.124) using a small to moderate effect size of f
= 0.15, α = 0.05, and β = 0.80, determined that a study sample of 55 participants would be sufficient to conduct multiple regressions.
All variables were determined to be normally distributed according to a Shapiro-Wilk test, as well as visual inspection of histograms and Q-Q plots. Initial Pearson’s bivariate correlations were conducted to determine relationships between 6MWT (i.e., distance walked), demographic factors (age, sex, household income), adiposity (i.e., VAT), diet quality (Total HEI-2015), ECAD™, and accuracy and reaction time during the flanker and hearts and flowers tasks. Subsequently, multiple hierarchical linear regression analyses were used to test the variance explained in cognitive outcomes by total distance walked during the 6MWT, following adjustment of the aforementioned demographic and adiposity variables. The significance of the change in the R2
value between the 2 steps was used to judge the independent contribution of distance walked during the 6MWT to variance in cognitive task performance, beyond that of step 1 or covariates. Given previous work among older children indicating that acute exercise has immediate post-exercise benefits for cognitive control, the task performance was compared based on the day of administration, i.e., visit 1 (after 6MWT) or visit 3 to account for any influence of the 6MWT on the hearts and flowers task performance. Statistical significance was set at p =
0.05. Statistical analyses were conducted in SPSS version 24 (IBM, Chicago, IL, USA). Given the abundance of literature among older children indicating a positive relationship between cardiorespiratory fitness and cognitive control (for review see [1
]), we employed one-tailed statistical tests, consistent with the directional hypothesis.
ERP subsample analysis: Within the subsample of children who completed the auditory oddball task, the relationship between physical fitness and the N2 and P3 components of the ERP waveform was assessed. The relationship with fat (VAT) was not assessed, as the inclusion of the VAT variable would further decrease the size of the subsample. Rather, the relationships between the neuro-cognitive indices, 6MWT performance, and demographic variables were explored via Pearson’s bivariate correlations, with subsequent partial correlations controlling for only age and sex, the two most salient demographic characteristics. Due to the directional nature of the ERP hypotheses, a p-value of 0.10 was used as a cutoff to determine moderate statistical relationships.