**4. Discussion**

We compared inter-limb coordination measures between TD children and children with ASD, while performing jumping jacks with and without rhythmic signal. Our main findings show no e ffect of the rhythmic signal on the coordination measures. However, children with ASD exhibited high variability in limb coordination, as shown by the LV measure, compared to the TD children. This is the first study to report a di fference in an inter-limb coordination measure between children with ASD and TD children, performing a simple, quick four-limb jumping activity.

While the maximal value of the limb accelerations was not a primary outcome measure of this study, we report lower accelerations in the ASD group compared to the TD group (statistically significant for the left leg and arm during self-selected rhythm jumps and not significant but showing the same trend for the other limbs and the rhythmic signal condition). This finding is expected, as several publications report slower repetitive hand and foot movement assessed with standardized test batteries in individuals with ASD, as reviewed by Gowen and Hamilton [22]. Also, drumming movements of children with ASD were reported as slower compare to TD children [23].

There was no statistically significant main e ffect of the rhythmic signal, provided during the jumping activity. Since there are reports of impaired early auditory pathways in ASD [24], the timing of motor neuron transmission in our ASD group may have been influenced by delayed auditory processing, rendering the cues unhelpful or even disturbing to the task execution. This explanation was suggested in a study that compared the cadence during an auditory-cued two-legged hopping task between TD and ASD groups [25]. While the TD group showed a high performance of synchronizing their jumps with the cues, the ASD group showed a varied deviant response to the cueing [25]. In·our study, however, we did not test for synchronization between the rhythmic signal and the movements of the children. Therefore, we cannot attest to the success or failure of the metronome in regulating the jumping sequence. Our results sugges<sup>t</sup> that the rhythmic signal has no e ffect on the inter-limb coordination or its variability between jumps in children (with or without ASD) while performing jumping jacks.

Surprisingly, we found no statistically significant di fferences in the TL outcome measure between the two groups. This finding suggests similar inter-limb coordination between TD and ASD children performing jumping jacks. We assume that this finding can be explained by the simplicity of the chosen activity. Contrarily to marching or drumming activities, which involve out-of-phase inter-limb movement, jumping jacks comprise of in-phase symmetrical limb movements. It was suggested that this type of activity produces a simultaneous activation of homologous muscle groups [26]. Therefore, the complexity of motor planning required for the activity chosen for this study might be smaller compared to activities such as gait. We assume that the deficits in motor planning in children with ASD contributed to the inter-limb coordination deficits in the studies reported in the literature due to the more complex task chosen. Conversely, for the jumping jacks activity, we surmise that the main factor influencing coordination is not motor planning, but the sense of proprioception. The ability to perform inter-limb coordinated movements relies, among other factors, on an intact sense of proprioception [27]. It has been reported that the sensory input of individuals with ASD is intact. Specifically, studies demonstrated no deficit in proprioception in individuals with ASD. For example, the accuracy and precision of the proprioceptive estimates of identifying the angle of the elbow and the position of the fingertip in adolescents with ASD was similar to adolescents without ASD [28]. We therefore conclude that the similarity in the TL between the ASD and TD groups relates to the characteristics of the jumping activity, selected for this study, which relies more on the sense of proprioception then on motor planning.

As expected, children with ASD exhibited high variability in limb coordination, i.e., high LV measures, compared to the TD children. This means that while the inter-limb asynchronization in TD children was consistent across consecutive jumps, the inter-limb asynchronization in children with ASD varied between consecutive jumps, and this di fference was statistically significant. High intra-individual variability is considered a marker for ASD [29,30]. The high intra-individual variability in ASD was

demonstrated for measures such as reaction time [31,32], hand grip strength [29], finger tapping [29], drumming [23], and walking tasks [29,33,34]. Our research makes an important contribution to the literature as it demonstrates the ability to perform objective quantitative discrimination between TD children and children with ASD using a simple and quick protocol of a four-limb activity. This protocol could be used in future studies to investigate di fferences in intra-subject variability between groups of children with ASD of di fferent sex, age, and level of IQ.

The main limitation of this study is the small sample size of the ASD group. Future studies should be encouraged by our preliminary results and continue the investigation on a larger population of children with ASD. Another limitation concerns the placement of the sensors, attached to the wrist and ankles of the subject. Although there were no between-group di fferences in BMI, slight variability of limb length between the children is expected. Accordingly, the maximal acceleration values may have been influenced by this, so that higher acceleration values would be measured when the sensor is located further from the shoulder or hip joint. This limitation, however, has no e ffect on the values of the TL and the LV since the calculations of these measures consider the di fference between both limbs. Also, the metronome frequency, set to 1 Hz, might not have been suited to all participants and they might have ignored it. Finally, although we report coordination deficits in children with ASD, these could be attributed to di fferences in IQ, motivation, or imitation ability.

In conclusion, our preliminary findings sugges<sup>t</sup> that the simple protocol presented in this study might allow an objective and accurate quantification of the intra-subject variability of children with ASD via actigraphy. This method should be further explored to discern between groups of children with ASD and other populations with motor dysfunction.

**Author Contributions:** Conceptualization, S.P.; methodology, S.P., L.V.G. and S.S.; software, S.P.; formal analysis, S.P.; investigation, S.S. and L.V.G.; resources, S.S. and L.V.G.; data curation, S.S. and L.V.G.; writing—original draft preparation, S.P.; writing—review and editing, S.P., S.S. and L.V.G.; visualization, S.P.; supervision, S.P.; project administration, S.P. and L.V.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors greatly appreciate the work of the following undergraduate occupational therapy students in data collection: T.S., N.I., B.L., and L.S.

**Conflicts of Interest:** The authors declare no conflict of interest.
