Effects of short-term arm immobilization on motor skill acquisition

Erin M King; Lauren L Edwards; Michael R Borich

doi:10.1371/journal.pone.0276060

. 2022 Oct 14;17(10):e0276060. doi: 10.1371/journal.pone.0276060

Effects of short-term arm immobilization on motor skill acquisition

Erin M King ^1,², Lauren L Edwards ², Michael R Borich ^2,^3,^*

Editor: François Tremblay⁴

PMCID: PMC9565666 PMID: 36240219

Abstract

Learning to sequence movements is necessary for skillful interaction with the environment. Neuroplasticity, particularly long-term potentiation (LTP), within sensorimotor networks underlies the acquisition of motor skill. Short-term immobilization of the arm, even less than 12 hours, can reduce corticospinal excitability and increase the capacity for LTP-like plasticity within the contralateral primary motor cortex. However, it is still unclear whether short-term immobilization influences motor skill acquisition. The current study aimed to evaluate the effect of short-term arm immobilization on implicit, sequence-specific motor skill acquisition using a modified Serial Reaction Time Task (SRTT). Twenty young, neurotypical adults underwent a single SRTT training session after six hours of immobilization of the non-dominant arm or an equivalent period of no immobilization. Our results demonstrated that participants improved SRTT performance overall after training, but there was no evidence of an effect of immobilization prior to task training on performance improvement. Further, improvements on the SRTT were not sequence-specific. Taken together, motor skill acquisition for sequential, individuated finger movements improved following training but the effect of six hours of immobilization was difficult to discern.

1. Introduction

Learning to coordinate motor sequences is essential for completing tasks necessary for daily living, such as tying a shoe or typing. Experience is a potent driver of neuroplasticity throughout the cortex [1], and even short-term experiences, such as motor skill practice, have been shown to induce structural and functional changes within the human motor cortex and to underlie motor learning [2, 3]. Synaptic processes such as long-term potentiation (LTP) and long-term depression (LTD) [4], alterations of dendritic spine density and morphology [5], and changes in inhibitory neurotransmission [3, 6] have all been shown to contribute to the training-induced plasticity that underlies motor skill acquisition.

Previous research in humans has demonstrated that experience-dependent synaptic strengthening through LTP-like plasticity in sensorimotor circuits is necessary for acquisition of motor skill [3]. However, in order to maintain stable levels of activity in these circuits, it is necessary to regulate synaptic strength at the level of the individual synapse to maintain synaptic homeostasis [7]. The model of homeostatic plasticity suggests that the degree of experience-dependent strengthening or weakening that can occur in a given synapse is influenced by the recent history of synaptic activity [8, 9], which prevents the circuit from becoming over- or under-excited [1, 10–12]. For example, in a synapse that has recently undergone a period of synaptic strengthening, additional synaptic strengthening becomes less likely to occur, and synaptic weakening becomes more likely. This principle has been demonstrated in both excitatory and inhibitory circuits within M1 using noninvasive brain stimulation (NIBS), where increasing activity of these circuits prior to plasticity-induction protocols resulted in reduced potential for further LTP-like plasticity [13, 14]. Since LTP is a primary contributor to experience-dependent plasticity, inducing LTD-like plasticity within M1 prior to training may leverage homeostatic mechanisms to enhance the capacity for task-specific synaptic strengthening and performance improvement in humans.

Short-term (≤12hr) limb immobilization transiently reduces sensory input to and motor output from the contralateral sensorimotor cortex, resulting in a temporary decrease in corticospinal excitability [15, 16], thought to reflect a decrease in synaptic strength through LTD-like processes [17], as well as EEG markers of synaptic potentiation [17]. Further, the capacity for LTP-like plasticity is enhanced immediately after short-term immobilization in human primary motor cortex [15]. Although short-term limb immobilization modifies systems-level indices of synaptic strength in humans, the effect on motor skill training-induced plasticity is unclear. Given that plasticity within human M1 underlies sensorimotor skill learning [3, 9, 18], an intervention that has the potential to enhance the capacity for LTP-like plasticity in M1 may influence skill learning. While several studies have found that motor performance on a variety of tasks is decreased after short-term immobilization [16, 17, 19–24], only one has examined the effect of immobilization on skill acquisition [22]. A study by Opie and Evans found no clear effect of immobilization on training during a grooved pegboard task [22]. However, no studies have examined the effect of immobilization on a task that requires individuated, sequenced finger movements. The purpose of the current study was to evaluate the effects of short-term limb immobilization on implicit motor skill acquisition in young, healthy individuals. In this study, participants completed a single motor skill training session after a period of 6 hours with or without immobilization of the non-dominant arm. We hypothesized that if short-term limb immobilization increases the capacity for activity-dependent synaptic strengthening in the corresponding contralateral M1 representation, then greater motor skill acquisition would be observed with training that followed immobilization compared to training following an equivalent period of no immobilization.

2. Materials and methods

2.1 Study participants

21 healthy individuals (6 male) aged 18–35 (24.8 ± 4.7 years) participated in the current study spanning the morning and evening of one day. The age range was selected in order to reduce the influence of age on motor skill learning [25]. Inclusion criteria included (1) no history of movement impairment or neurodegenerative disease and (2) right handedness according to the Edinburgh Handedness Scale [26]. All study procedures were approved by the Emory University Institutional Review Board in accordance with the Declaration of Helsinki. Written consent was obtained from all participants prior to testing procedures. Behavioral data from one participant were excluded due to equipment malfunction.

2.2 Motor task paradigm

A version of the Serial Reaction Time Task (SRTT) [27, 28], an implicit motor task, modified from Clos and Sommer was used in this study [29]. Participants placed the fingers of their left (non-dominant) hand on a custom-made button box. The buttons on the box corresponded to the top four colored squares on a computer display placed at eye-level in front of the participant (Fig 1). Participants were instructed to press the button corresponding to the top square that matched the color of the bottom (target) square as quickly and accurately as possible. An example trial and overall study design are shown in Fig 1. In the morning baseline (BL) session, participants completed one test block that consisted of 280 button presses (50 random, 180 repeated, 50 random) during the session prior to the immobilization period to assess motor performance [30, 31]. Repeated button presses consisted of 15 repeats of a 12-item sequence, and the participants were not informed there was a repeating sequence.

Fig 1 — (A) Participants were instructed to press the key on a custom button box that corresponded to the top square that matched the target (bottom) square (B) Sequence-specific skill (SKILL) was calculated as the mean difference in response time (RT) between repeated (white) and random (gray) sequences for test blocks at each time point.

2.3 Arm immobilization

After completion of the BL session, 10 participants were randomly assigned to undergo arm immobilization. These individuals were instructed to wear a finger control mitt on the left (non-dominant) hand, which secured positioning of the fingers in a padded mitt to restrict finger movement. In addition to the hand mitt, the arm was placed in a sling to reduce movement of the wrist, elbow, and shoulder joints. Participants were instructed to move their left arm as little as possible during the immobilization period but to use the non-immobilized right (dominant) hand and arm normally. Individuals in the control group (n = 10) were instructed to use both arms normally between sessions.

2.4 Motor skill acquisition paradigm

After the 6-hour immobilization period, skill acquisition was assessed in the evening using a pre-train test block (PRE), five training blocks consisting of 180 repeated button presses, and a post-train test block (POST). Colors changed each trial in order to mask the sequence and prevent explicit awareness of the sequence. At the end of the evening session, the degree of explicit awareness of the presence of a sequence was assessed by asking if the participants noticed any pattern of button presses during the task. If they indicated that they noticed a pattern during training, they were asked to freely recall the sequence. The ability to freely recall ≥4 consecutive items of the 12-item sequence was considered explicit awareness [30, 31].

Raw data files as well as a summary data file can be found here: https://osf.io/wazdy/?view_only=cfed7648613f45589c86d73582f93ce7

2.5 Behavioral outcome measures

During the six-hour immobilization period, activity monitors (wGT3x-BT, ActiGraph) were worn on both arms by all participants to determine compliance with the immobilization procedure since participants were allowed to leave the lab between test sessions. Movement of each arm, measured in units of Gs, was collected bilaterally (left/target and right/non-target arms) throughout the six-hour immobilization period for both groups. A two-way ANOVA was performed to examine the effect of immobilization on activity counts, with within-subject factor of hand (two levels: target and non-target) and between-subject factor of group (two levels: immobilization or control group).

2.5.1 Assessment of general motor performance

Response time for each button press was acquired during task performance with a custom Java script. Outlier response times, defined as a response time three standard deviations greater than the mean response time within each block for each participant, were removed from analysis.

General motor performance was assessed by calculating the response times for button presses across task exposure. Response times were then normalized to the average response time for the first 50 random button presses in order to account for variations in baseline motor performance. Normalized response times for random sequence in the test blocks and repeated sequence in the training blocks were analyzed separately. A two-way ANOVA was used to assess the effect of immobilization on general motor performance, as measured by the normalized response time for the first 50 random sequence button presses during each of the three test blocks, with within-subject factor of time (three levels: baseline (BL), pre-training (PRE), and post-training (POST)) and between-subject factor of group (two levels: immobilization or control group). A separate two-way ANOVA was used to assess normalized response time for repeated sequence performance across the training blocks (five levels: Training blocks 1–5) with between-subject factor of group (two levels: immobilization or control group).

2.5.2 Assessment of sequence-specific skill

To assess sequence-specific skill, two outcome measures were calculated: Skill Score (SS) and Interference Score (IS). In line with previous studies using a similar version of the SRTT and task design [30, 31], the Skill Score (SS) was calculated as the average response time for the last 50 random button presses of each test block minus the average response time for the last four repeated sequences (48 repeated button presses) preceding the random presses [32]. This was kept consistent across test blocks to control for potential order effects. A larger SS indicates greater sequence-specific skill, such that when trained, participants’ repeated sequence performance is faster than random sequence performance. Skill Scores were calculated for BL (SS_BL), PRE (SS_PRE), and POST (SS_POST) test blocks.

The Interference Score (IS) was calculated to assess potential interference caused by an abrupt transition from repeated sequence to random sequence (Rep-Rand) button presses, which leads to an increase in response time [28]. Rather than assessing the relative response times of repeated sequence and random sequence button presses averaged over many button presses, the IS examines the impact of disrupting the trained motor sequence with a random sequence by calculating the average response time for the first 12 random button presses immediately following a transition minus the average response time for the 12 repeated button presses immediately preceding the transition. A larger IS represents a larger increase in response time at the transition from random to repeat and therefore greater interference. Interference Score (Rep-Rand) was calculated for each test block (IS_BL, IS_PRE, IS_POST).

2.5.3 Task performance of the non-immobilized hand

Previous research has demonstrated that interhemispheric interactions between motor cortices have been shown to change after a period of immobilization [33]. In order to assess possible effects of immobilization of the left hand on the right hand, two test blocks of the SRTT were performed with the right, nontarget hand: one at the BL timepoint and one at the PRE timepoint. Statistics were performed in order to assess the effects of immobilization on both general motor performance as well as sequence-specific skill, although no training blocks occurred between these two test blocks. A two-way ANOVA was used to examine normalized response time for the last 50 random button presses of each test block. A separate two-way ANOVA was used to assess skill score before and after the immobilization period (two levels: BL and PRE blocks) with between-subject factor of group (two levels: immobilization or control group).

All statistical analyses were performed with Prism GraphPad 8, and a critical α was set at 0.05 corrected for multiple comparisons as appropriate. Normality and homogeneity of variance were statistically confirmed with the Shapiro-Wilk’s Test, descriptive statistics, and Levene’s Test.

3. Results

A two-way ANOVA demonstrated that there were significant effects of time (F = 60.1, p < .0001) and of group (F = 14.3, p < .01), as well as a time X group interaction (F = 40.5, p < .0001) on average activity counts (Fig 2). Sidak’s multiple comparisons test indicated that activity in the immobilized (target) arm were significantly reduced in immobilized individuals (t = 9.98, p < .0001) confirming participants complied with the immobilization procedure. Table 1 summarizes the ANOVA results for activity counts.

Fig 2 — Participants wore activity monitors on both wrists throughout the six-hour immobilization period. Activity counts in the immobilized (target) arm were significantly reduced compared to the non-immobilized (nontarget) limb in immobilized participants (t = 9.98, p < .0001).

Table 1. Activity counts ANOVA results.

Measure	Source	DFn, DFd	F Statistic	p-value	Effect size (eta²)
Activity Counts	Time	1, 18	60.1	< .0001	.223
	Group	1, 18	14.3	< .01	.248
	Time X Group	1, 18	40.5	< .0001	.151

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Significant p-values are bolded.

3.1 Assessment of general motor performance

Overall, 1.99% of button presses were removed (694/34,800 total button presses) across all participants (range: 1.38%-2.76%). Average response times were determined to be normally distributed for each block of button presses: BL (W = .95, p = .3), PRE (W = .95, p = .4), TRAIN1 (W = .95, p = .34), TRAIN2 (W = .95, p = .38), TRAIN3 (W = .97, p = .74), TRAIN4 (W = .97, p = .78), TRAIN5 (W = .94, p = .21), and POST (W = .98, p = .95). Our results demonstrate that general motor performance improved across task exposure, measured by a decrease in response time for both random and repeated button presses with task exposure. The overall accuracy for control participants was 97.4%, and the overall accuracy for immobilized participants was 96.4%. Normalized response times for each group across training are shown in Fig 3.

Fig 3 — Values less than one (black dashed line) indicate faster than baseline performance. Open circles represent 50 random button presses, and closed circles represent 180 sequenced button presses. One test block occurred in the morning (BL Test) to assess baseline motor performance. The evening session consisted of five training blocks (Training) with test blocks before (PRE Test) and after (POST Test). Error bars represent standard error.

Random sequence performance increased across test blocks, as measured by a decrease in response time for the last 50 random button presses of each test block, for both control and immobilized participants. A two-way ANOVA indicated that there was a significant main effect of time (F = 57.5, p < .0001) (Fig 4A) on normalized response time; however, there was not a main effect of group nor a time X group interaction.

Fig 4 — Normalized response time significantly decreased across training regardless of immobilization condition (F = 57.5, p < .0001). *p < .05, **p < .01, ***p < .001, ****p < .0001.

A two-way ANOVA showed a significant main effect of time (F = 6.3, p = .0008) on normalized response time for repeated sequence performance across training blocks 1–5 (Fig 4B). There was no effect of group nor a time X group interaction on response time throughout training blocks.

3.2 Assessment of sequence-specific skill

Skill score was demonstrated to be normally distributed in the PRE (W = .97, p = .68) and POST (W = .92, p = .11) blocks but not at baseline (W = .83, p = .002). Further assessment using descriptive statistics did not show substantial violations of the assumptions of normality or homogeneity of variance. Interference score was normally distributed at BL (W = .97, p = .85), PRE (W = .96, p = .54), and POST (W = .98, p = .86) timepoints.

Both groups showed an average increase from SS_BL to SS_PRE, but a two-way ANOVA showed no effect of group or time on skill score and no time X group interaction (Fig 5A) across all three timepoints. Similarly, a two-way ANOVA showed no effect of group or time nor a time X group interaction on interference scores. Fig 5B shows the interference scores for the transition from repeated to random for BL, PRE, and POST test blocks. Table 2 summarizes the results of each ANOVA for the primary SRTT-based outcome measures.

Fig 5 — Neither (A) Skill Score nor (B) Interference score significantly changed across task exposure for either group.

Table 2. SRTT ANOVA results.

Measure	Source	DFn, DFd	F Statistic	p-value	Effect size (eta²)
Random Sequence Performance	Time	1.8, 32.6	57.5	< .0001	.570
	Group	1, 18	0.16	.69	.002
	Time X Group	2, 36	1.4	.25	.014
Repeated Sequence Performance	Time	3.1, 56.5	6.3	.0008	.026
	Group	1, 18	0.44	.51	.022
	Time X Group	4, 72	0.19	.95	.0007
Skill Score	Time	1.6, 29.6	0.73	.46	.023
	Group	1, 18	0.30	.59	.007
	Time X Group	2, 36	0.12	.89	.0034
Interference Score	Time	1.3, 23.3	0.03	.91	.001
	Group	1, 18	0.24	.63	.005
	Time X Group	2, 36	0.46	.64	.016

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Significant p-values are bolded.

3.3 Task performance of the non-immobilized hand

Response times for the right, non-immobilized hand were determined to be normally distributed in both BL (W = .96, p = .47) and PRE (W = .94, p = .25) blocks. A two-way ANOVA demonstrated that normalized response time decreased from BL to PRE timepoints, with a main effect of time (F = 103.2, p < .0001). There was no effect of group, nor a time x group interaction (Fig 6A).

Fig 6 — (A) Response time decreased from BL to PRE timepoints across participants (F = 103.2, p < .0001), but was not immobilization-specific. (B) Skill score did not significantly change from BL to PRE timepoints across groups.

Skill score was normally distributed for BL (W = .97, p = .74) and PRE (W = .96, p = .49) blocks. A two-way ANOVA showed no effect of time nor group on skill score, nor a time x group interaction (Fig 6B). Full ANOVA results can be found in Table 3.

Table 3. Nontarget hand motor performance.

Measure	Source	DFn, DFd	F Statistic	p-value	Effect size (eta²)
Random Sequence Performance	Time	1, 18	574.0	< .0001	.93
	Group	1, 18	2.9	.11	.005
	Time X Group	1, 18	2.9	.11	.005
Skill Score	Time	1, 18	.67	.42	.016
	Group	1, 18	.04	.85	.001
	Time X Group	1, 18	2.7	.12	.066

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Significant p-values are bolded.

4. Discussion

In the current study, we investigated the effect of short-term (6 hours) arm immobilization on implicit motor sequence acquisition. General motor performance improved with task exposure in both groups; however, improvement was not sequence-specific. Despite confirming that the immobilization protocol was followed by individuals in the immobilization group, no group differences were found in general motor performance or sequence-specific skill. Overall, our results suggest that immobilization did not significantly augment implicit, sequence-specific skill acquisition or online improvements in general motor performance on the SRTT.

4.1 Lack of an effect of immobilization on motor performance on an individuated finger sequencing task

Contrary to the results of previous studies utilizing short-term immobilization [16, 17, 19–24], we did not observe a decrement in motor performance after a period of upper limb immobilization. In fact, general motor performance increased from BL to PRE test blocks in both groups. One potential explanation for the difference in our results could be the duration of immobilization. While changes in measures of corticospinal excitability have been shown in as little as 3 hours after onset of immobilization [16], most previous studies that found impaired motor performance after immobilization used immobilization periods of at least 8 hours. In the current study, arm immobilization occurred for a period of six hours. The idea that duration of immobilization could influence motor performance is supported by a study by Moisello and Bove, which found that motor performance on an out-and-back cursor task decreased after 12, but not 6 hours, of immobilization [21]. Therefore, it is possible that a greater duration of immobilization may be necessary to affect motor performance. Alternatively, limb immobilization may have a greater behavioral effect on tasks that require multi-joint coordination of the entire limb (e.g., skilled reaching [17]) rather than individual digits in the hand required to perform the SRTT in the current study.

4.2 Acquisition of sequential, individuated finger movements was not preferentially enhanced after immobilization

Despite the potential for short-term immobilization to enhance experience-dependent plasticity within M1, our results demonstrated that implicit motor skill acquisition on a task that requires coordination of sequential movements was not influenced by immobilization. While M1 has been shown to be involved in the execution of sequential movements [34, 35], it is unclear whether M1 has a role in acquisition of the sequenced motor skill itself [36]. Therefore, increasing the capacity for LTP-like plasticity in M1 may not be sufficient to influence acquisition of an implicit, sequenced motor skill. Outside of M1, short-term immobilization has been shown to influence markers of plasticity in primary somatosensory cortex [17], but it is unclear whether immobilization influences plasticity in other brain areas that may be involved in acquisition of sequence-specific skill, such as premotor cortex or supplementary motor area [36].

4.3 Immobilization of the non-dominant limb did not influence dominant limb motor performance

Even though short-term immobilization has been shown to influence interhemispheric interactions [33], our results demonstrated no evidence of an effect of six hours of immobilization on motor performance of the non-immobilized right hand. Results from previous studies have demonstrated improved performance of the non-immobilized hand on motor tasks after longer periods of immobilization, such as one [37] or two weeks [38]. Behavioral effects of immobilization may differ based on the duration, as longer durations of immobilization (days to weeks) have been shown to lead to additional changes in spinal excitability [39, 40] and cortical morphology [38] that have not been previously observed with shorter durations (6 hours) of immobilization. It is possible that effects of immobilization on the motor performance of the non-immobilized limb is also dependent on the duration of immobilization, and we would predict a similar finding of increased performance of the non-immobilized hand with longer periods of immobilization.

4.4 Task characteristics may influence the effects of immobilization

One unique aspect of the current study was that this was the first study to examine the effect of immobilization on a sequence learning task that relies on individuated finger movements. Therefore, the different characteristics of the task itself as well as the outcome measures could have contributed to inconsistent results between this study and others. Several previous studies that observed a decrease in motor performance after a period of immobilization used tasks that required control of more proximal portions of the upper extremity, such as reaching, while the current study used a modified SRTT that emphasized fine control of the distal upper extremity. Previous research has demonstrated that the composition of descending projections to the distal and proximal upper extremity are different in primates [41–43], and it is possible that immobilization differentially modulates these pathways. Bolzoni and Bruttini suggested that function of the proximal muscles responsible for postural control is more likely to be influenced by a period of immobilization, even when only distal hand muscles are immobilized [20]. Similarly, in a task requiring participants to pick up and put down a pencil repeatedly, immobilization of the dominant arm increased reach duration and changed acceleration and deceleration of movement but did not influence grip aperture [19]. In the current study, outcome measures to assess general motor performance and sequence-specific skill were calculated using response time, which are not able detect changes in joint kinematics that may occur after a period of immobilization. Future studies can quantify joint coordination during task performance with kinematic data [44] and/or separating response time into reaction time and movement time to examine central nervous system contributions to changes in SRTT performance with training and immobilization [45].

Another potential explanation for the observed findings is that immobilization impairs proprioceptive processing, and tasks that require proprioceptive information to complete will be impacted by a period of immobilization. This idea is supported by Avanzino and Pelosin that showed that neurophysiological changes normally seen after immobilization were blocked when proprioceptive receptors were selectively activated during the immobilization period [46]. This could explain why performance on the modified SRTT, which required small amplitude, individuated finger movements that would not be expected to be affected by postural control or modulation of proprioceptive receptor activity, was not negatively impacted by immobilization. Taken together, our findings support prior literature suggesting that multi-joint coordination of arm movements may be preferentially impacted by upper limb immobilization. It remains unclear if immobilization can modulate the acquisition of skill for tasks requiring multi-joint coordination of the arm.

4.5 Skill improvements across groups were not sequence-specific

An unexpected result from the current study was that sequence-specific skill did not significantly improve after training. One possible explanation for the lack of change in skill score and interference score in across training is that time of day influenced sequence-specific skill acquisition, since all motor training was performed in the evening session for the current group of participants in our study. Previous research has suggested that skill improvement on a sequence learning task is greater in the morning compared to the evening [47], which does not seem to be the case with acquisition of skill in a repetitive ballistic motor training task [48]. Interestingly, Keisler and Ashe suggested that motor sequence learning itself may not be impaired in the evening relative to morning, but factors such as motivation, attention, and fatigue may lead to the impairment of the expression of learning (in the form of task performance) [49]. Additionally, previous research has shown that performance during a skill acquisition task cannot be equated with skill learning. In fact, certain features of a motor task itself, such as task difficulty or practice structure, can lead to a decrease in performance during the acquisition phase of learning but subsequently enhance retention of skill during a follow-up assessment [50, 51]. Including delayed retention testing could assess the effect of immobilization on motor sequence learning when training occurs in the evening.

4.6 Study limitations

There are several limitations to the current study. The current study only assessed within-session skill acquisition, thus, the effect of upper limb immobilization on skill learning remains unknown. Skill retention and generalization can be evaluated in future studies to determine if immobilization has an effect on skill learning. A priori sample size calculations were based on pilot work and previously published studies showing large effect sizes of immobilization on motor performance. Although the effect of immobilization on motor skill acquisition in the current study was consistent with the hypothesized direction, the observed effects sizes were small. Future studies with larger sample sizes can be conducted to detect small effect sizes, if present, or to test equivalence. Additionally, it is possible that the color-matching component of the task may have masked the sequence, making sequence-specific acquisition, even implicitly, more difficult that could have contributed to the lack of change in sequence-specific skill after a single training session. Future studies could employ different versions of the SRTT to address this limitation or investigate other tasks more closely aligned with the effects of arm immobilization (e.g., skilled reaching movements).

5. Conclusions

Overall, our results suggest that short-term (6 hours) immobilization of the arm has a small effect on implicit skill acquisition on a task that requires individuated, sequenced finger movements. However, it is possible that task characteristics and the duration of immobilization influenced the results. These initial findings suggest the behavioral effects of short-term arm immobilization may be task specific and depend on duration of immobilization. Future studies should assess the effects of immobilization on skill acquisition and learning using tasks that require multi-joint control and/or proprioceptive feedback to understand the capacity for immobilization to augment endogenous experience-dependent plasticity associated with training or task-specific rehabilitation.

Acknowledgments

The authors would like to acknowledge Scott Heston for his contributions to programming the motor task and creating the button boxes used during the task, as well as Maria Krakovski and Martin Tan for their assistance with data collection and processing.

Data Availability

Raw behavioral data are available from the Open Science Framework database (link to view: https://osf.io/wazdy/?view_only=cfed7648613f45589c86d73582f93ce7).

Funding Statement

EMK is supported by the Emory Mechanisms of Learning Across Development and Species Training Grant 2T32HD071845-06 (https://sites.google.com/view/mechanismsoflearning/home), a Ruth L. Kirschstein Institutional National Research Service Award through the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0276060.r001

Decision Letter 0

Benjamin A Philip

20 Sep 2021

PONE-D-21-19679Effects of short-term arm immobilization on motor skill acquisitionPLOS ONE

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Reviewer #1: In the current study, the authors investigated the influence of plasticity mechanisms via limb immobilization on implicit skill acquisition. This work is theoretically driven and generally well written. Both control and immobilized groups improved the random and repeated sequences, without any difference between the groups.

While this topic is of interest, I have general and specific concerns that limit the value of the current findings.

General remarks:

- Even if it is clear and well written, the introduction focuses a lot on neurophysiogical aspects of immobilization. Considering, however, this research field as well as the used experimental paradigm, I personally think that the introduction section lacks of a paragraph that focuses on behavioral results. To present behavioral literature at the end of the introduction would give a clearer overview of the state of the art and to build a bridge with the method section.

- Also, given the introduction, as well as the lines 79-80, one would expect neurophysiological measurements to support the behavioral predictions (e.g., to check if corticospinal excitability decreased after immobilization). Also, if there is no method nor results for transcranial magnetic stimulation, why is the contraindication to TMS an exclusion criterion? Unfortunately, this could suggest that TMS has been used, but that the results were not reported, raising question about the reasons that could have motivated such decision.

- The main conclusion is that short-term arm immobilization does not modulate motor acquisition in SRTT. First, and as stated by the authors themselves, quite comparable results have been also observed in Moisello et al. (2011). It seems that a minimum of 8-hour immobilization is required to induce behavioral changes. Could the authors clarify the rationale for the 6-hour immobilization? Also, based on the current analysis, the claim that short-term immobilization does not modulate motor acquisition, and that “Acquisition of sequential, individuated finger movements is not preferentially enhanced” cannot be stated in such way. One cannot conclude on the absence on an effect with such statistics. For that purpose, I recommend to perform specific statistics, such as equivalence testings (see Lakens et al., 2018).

- Could the author clarify the experimental design? What was the purpose of performing the repeated sequence between 2 blocks of random sequences? What is the goal of the second block of random sequence?

- Could the authors specify in the abstract and/or the conclusion, when missing, the duration of the immobilization (which seems to be important to induce behavioral changes) and that it was an implicit sequence task?

Specific comments:

Abstract

Line 25: I would suggest to remove “younger”, or to replace it by “young”.

Methods

Lines 136-137: Could you please provide information about which data proportion have been removed? And were trials with RT below 100ms removed?

Line 142: Has the normality been verified prior to the analyses?

Lines 151: Sequence-specific skill.

- Skill score: 48 button presses of repeated sequence (at baseline, pre and post) minus the 50 button presses of the last random sequence at baseline. Why did the authors choose the last random sequence at baseline?

Line 170: Please provide here information about which post-hocs were used.

Results

Please provide effect sizes and the full degrees of freedom for each reported Anovas.

It may be a personal misunderstanding, but I do not understand why the post hocs are reported with the letters "t" or "q".

l.197: results of random sequence performance. The authors did not show any statistical results for this outcome in the text. It seems we have to wait for Table 1 to see the results. Please add information in the text for clarity. How is the statistical design for the random sequences? Did the authors merge the first and the second block of each test? Or was it integrated as a specific factor within the ANOVA?

l.201-205: As there was only a main effect of Time, it is not relevant to show separate comparisons for each group.

l.213: As there was only a main effect of Time for the training data analysis, it is not accurate to perform post-hoc tests for each group individually (“the normalized response time for training block 5 was significantly faster than training block 1 in the immobilized participants (t=4.2, p=.024) but not the control participants (t=2.5, p=.30)”).

About the results of the training block, how did the authors normalize data of the repeated sequences? In Figure 2 (with all the blocks and sequences), it seems that 1) RT of the last 3 blocks of the training were shorter than RT at post for the repeated sequence and 2) RT for the immobilized group are shorter, especially at block 5, in comparison to the control group.

Discussion

The authors should revise the discussion based on the appropriate statistics.

Figure and Tables

Figures 2, 3 and 4 are intermixed in the text.

l.179: “Fig 2. Immobilized participants complied with the immobilization procedure”.

l.187: “Normalized response times for each group across training are shown in Figure 3”.

In Table 1, for the repeated sequence performance, what data are presented? The raw data or the normalized data?

Reviewer #2: King and colleagues investigated whether short-term arm immobilization influences motor skill acquisition by employing a modified Serial Reaction Time Task (SRTT) in healthy adults. The motor skill acquisition was assessed after six hours of immobilization of the non-dominant arm (experimental group) or an equivalent period of no immobilization (control group). Results showed an improved performance overall after training, not influenced by immobilization. The authors conclude that six-hour immobilization does not augment motor skill acquisition for sequential, individuated finger movements.

The topic addressed in this paper is relevant. The manuscript is overall nicely written and easy to follow. However, before publication, I have a few general suggestions that might be worth considering in the introduction and discussion and a few minor suggestions regarding the methods.

Introduction

The introduction contains a detailed description of the studies that have investigated the effect of limb immobilization on synaptic processes, long-term potentiation, and long-term depression. Whereas this background can give a broad idea of limb immobilization, it does not provide the rationale for this study. It is not clear why we should study short-term limb immobilization for motor skill training-induced plasticity, since it has already largely been investigated with different tasks (e.g., Moisello et al., 2008; Ngomo et al., 2012 https://doi.org/10.1016/j.neuroscience.2012.06.018; Opie et al., 2016; Lundbye Jensen et al, 2005 https://doi.org/10.1152/japplphysiol.01408.2004). What is the novelty of the present study?

Regarding limb immobilization, the authors only comment on studies that investigated the motor system with non-invasive brain stimulation techniques. However, it could be interesting to read a few words about the M1 activity after immobilization with different other methods (e.g., Huber et al. 2006 https://doi.org/10.1038/nn1758; Avanzino et al. 2011 https://doi.org/10.1523/JNEUROSCI.4893-10.2011; Garbarini et al., 2018 doi: 10.1093/cercor/bhy134; Langer et al. 2012 doi: 10.1212/WNL.0b013e31823fcd9c; and, in a similar vein Sperl et al., 2021 https://doi.org/10.1007/s00221-021-06190-w).

The authors predict that short-term limb immobilization would lead to a greater motor skill acquisition with training that followed immobilization, based on increased capacity for activity-dependent synaptic strengthening in the corresponding contralateral M1 representation. However, the opposite prediction can be hypothesized since studies found that even short limb immobilization affects motor performance (e.g., Moisello et al., 2008).

Methods

This study has a between-subjects design, with ten participants per group. In my opinion, this is a minimal sample size, is there a reason? If an a priori analysis about sample size was not performed, I think the authors should add this as a limitation of the study.

Another consideration is that probably an interesting control for this task should have been the no-immobilized hand, instead of a control group. In this way, the authors could have used a within-subjects design. What do expect to find if they had used the dominant hand as a control, in light of the literature about use-dependent hemispheric balance (see for example Avanzino et al., 2011 https://doi.org/10.1523/JNEUROSCI.4893-10.2011)?

Did the authors collect accuracy of the responses too?

Discussion

In a recent study on motor inhibition and limb immobilization (Bruno et al., 2020 https://doi.org/10.1016/j.neuroimage.2020.116911), the authors found no modulation of reaction times (RTs) in a Go/Nogo task between pre- and post immobilization (one week) of the non dominant hand. On the contrary, they found a better performance (lower RTs) for the dominant no-immobilized hand. Is this result in line with the findings of the present manuscript? Can di author comment on this?

The result about the better performance (lower RTs in train 5 as compared to train 1, figure 4B) in the immobilized group is not discussed. Can the authors spend a few words on this? In some way, this is a result suggesting that the immobilization induced a better motor skill.

Minor

Why the TMS requirements had to be respected? In this study, TMS was not employed.

Why did the authors choose a six-hour immobilization?

The name of the figures is wrong (figure 2 is called 3, and vice versa).

I did not find in the text the link with the raw data.

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PLoS One. 2022 Oct 14;17(10):e0276060. doi: 10.1371/journal.pone.0276060.r002

Author response to Decision Letter 0

17 Mar 2022

Response to Reviewers

While this topic is of interest, I have general and specific concerns that limit the value of the current findings.

Thank you for the positive and constructive comments regarding the original submission. We have extensively revised the manuscript based on the concerns raised to increase the value of the observed findings to the readership of PloS ONE.

General remarks:

- Even if it is clear and well written, the introduction focuses a lot on neurophysiogical aspects of immobilization. Considering, however, this research field as well as the used experimental paradigm, I personally think that the introduction section lacks a paragraph that focuses on behavioral results. To present behavioral literature at the end of the introduction would give a clearer overview of the state of the art and to build a bridge with the method section.

We appreciate this comment that increases the connection between the current project objective and the behavioral literature. Additional information has been added to the Introduction section (lines 70-77) to contextualize the current study with existing behavioral literature.

We appreciate identifying this oversight. This inclusion criterion was part of a separate experiment that included TMS delivery, and it has been removed from the revised submission for clarity.

Thank you for raising these important points. We selected a six-hour immobilization period both to increase feasibility for the experimental protocol based on findings of our pilot work and to support the likelihood of future translation to real-world and/or clinical settings. Given the literature, it is possible that at least 8hrs of immobilization is necessary for a behavioral effect. We have expanded on this point in the revised Discussion section (lines 379-383, 413-416).

We agree that it is not possible to determine the absence of an effect with the statistical analyses performed. The experimental design and a priori sample size calculation was not intended for formal equivalence/non-inferiority testing. We have softened the language in the Discussion section to clarify the interpretation of observed findings that there was a lack of evidence of an effect of six hours of immobilization on motor skill acquisition.

We have also revised the Limitations section in the Discussion to clarify that the findings observed were in the hypothesized direction, but the effect sizes were smaller than previous studies used to power the present study and the study was not powered to determine equivalence or to detect small effect sizes. Future studies with larger sample sizes could be performed to build upon the current findings (lines 489-497).

In line with previous studies utilizing a similar modified version of the SRTT (Robertson et al., 2004; Cohen et al., 2005), the test block has a “sandwich” design, consisting of repeated sequence button presses flanked on either side by random button presses. The skill score is derived from the last 48 repeated button presses (4 repeats of the 12-item sequence) and the second block of random sequence button presses. Having random trials before and after the block of repeated sequences at each time point provided a balanced test block design and offered the opportunity to compare our results to previous studies utilizing the SRTT. We have added references to the Methods (lines 107, 175-176)

Thank you for this request. The information has been added throughout.

Specific comments:

Abstract

Line 25: I would suggest to remove “younger”, or to replace it by “young”.

Thank you for noting this language which we usually use when evaluating group differences between ‘younger’ and ‘older’ adult cohorts. In the current study, we have revised to ‘young’ as suggested.

Methods

Lines 136-137: Could you please provide information about which data proportion have been removed? And were trials with RT below 100ms removed?

Overall, 1.99% of button presses were removed (694/34,800 total button presses) across all participants (range: 1.38%-2.76%). This information has been added to the text (lines 234-235). No response times were below 100ms, although that was not a criterion for removal. It was possible that implicit knowledge of the sequence could allow for the participants to press the correct button before processing the visual stimulus; however, evidence was not found for this effect in the current study.

Line 142: Has the normality been verified prior to the analyses?

Thank you for noting this point missing from the original manuscript. Normality was statistically assessed with the Shapiro-Wilk's test and inspection of data distribution and Q-Q plots to verify homogeneity of variance. This information has been added to the text in the Methods and Results sections.

Lines 151: Sequence-specific skill.

In line with previous studies using a similar version of the SRTT and task design (Robertson et al., 2004; Cohen et al., 2005), the skill score was calculated as the response time for the last 50 random button presses of each test block minus the 48 button presses of repeated sequence immediately preceding it. We have included this information in the text to make it clearer. The last random and repeated sequences were used in all test blocks in order to account for possible within-test block order effects.

Line 170: Please provide here information about which post-hocs were used.

Sidak’s multiple comparisons tests were used when a time x group interaction was found. This information has been added to the Results section (line 216).

Results

Please provide effect sizes and the full degrees of freedom for each reported Anovas.

Effect sizes (eta squared) and degrees of freedom have been added for all ANOVA tables.

It may be a personal misunderstanding, but I do not understand why the post hocs are reported with the letters "t" or "q".

Post-hoc t-tests have been removed (see below comments for context).

This information has been included and minor edits have been made for clarity. We only included the second set of random button presses in each test block in the analysis for general motor performance. We performed this analysis to evaluate general improvements in motor performance on the SRTT associated with both immobilization and training on the repeated sequences.

l.201-205: As there was only a main effect of Time, it is not relevant to show separate comparisons for each group.

Considering this comment, we have removed the separate comparisons for each group.

The post-hoc tests have been removed.

Average response times were all normalized to the first 50 random button presses in the Baseline test block. Response times do appear to have qualitatively increased from the end of training to the post-training block, which could have been due to the transition from repeated button presses to random button presses. Other alternatives include the short break (30 seconds to a minute) between the last training block and the post-test block leading to an increase in response time, or there could be an effect of fatigue. Mean RT was shorter for the immobilized group towards the end of training, but due to interindividual variability, the difference did not reach statistical significance.

Discussion

The authors should revise the discussion based on the appropriate statistics.

We have made several revisions to the Discussion section to more closely align with the results from the updated statistical analyses performed.

Figure and Tables

Figures 2, 3 and 4 are intermixed in the text.

l.179: “Fig 2. Immobilized participants complied with the immobilization procedure”.

l.187: “Normalized response times for each group across training are shown in Figure 3”.

The figure names have been updated to correct this issue identified

In Table 1, for the repeated sequence performance, what data are presented? The raw data or the normalized data?

The normalized data are provided in Table 1.

Thank you for the constructive review and positive remarks regarding the original submission. We have incorporated each suggestion into the revised manuscript to enhance the clarity and utility of the project findings

Introduction

Several studies have assessed the effect of immobilization on motor performance, but few have assessed the effect of immobilization on skill acquisition, and none have assessed the effect of immobilization on acquisition of skill requiring sequenced, individuated finger movements. Some context regarding how the current study fits in with existing studies of motor performance after immobilization has been added to the Introduction section (lines 70-77).

Thank you for this comment and opportunity to include additional relevant findings investigating M1 activity post-immobilization. We have added description of neurophysiological changes associated with immobilization using methods other than TMS in the Introduction (line 65) and Discussion (lines 407-409).

We agree that immobilization has been shown to affect performance of certain types of motor tasks including skilled reaching. Based on the previous literature demonstrating reduced motor performance after immobilization, we hypothesized that the ability to perform a serial key press task would be temporarily reduced after immobilization, but the ability to acquire sequence-specific motor skill would be enhanced based the concept of increasing the dynamic range of synaptic strengthening through immobilization. Investigating both performance on random and repeated sequences offered the opportunity to concomitantly evaluate the effects of immobilization on both general motor performance and sequence-specific skill acquisition. Although the observed effect sizes were small, the direction of effect was in line with the hypothesis that immobilization may increase the dynamic range for synaptic strengthening. We have clarified these points in the Introduction and Discussion sections.

Methods

An a priori sample size was performed based on previous immobilization studies and our pilot work. However, the effect sizes observed in previous published studies were large (e.g., Moisello et al., 2008) in comparison to the small effects observed in the current study. We have acknowledged the limited sample size considering the small effect sizes observed in the current study which has been added as a potential limitation in the Discussion section.

Thank you raising this important point. SRTT data from the right hand were collected before and after the immobilization period (BL and PRE test timepoints) to assess motor performance of the non-immobilized hand. Based on the results from Avanzino et al., 2011, we would have predicted an increase in performance of the non-immobilized hand after the immobilization period. However, we found no evidence of an effect of six hours of immobilization on the performance of the non-immobilized hand. This could be due to factors such as the duration of immobilization. We have added this analysis and interpretation to the revised manuscript.

Did the authors collect accuracy of the responses too?

Yes, accuracy data were collected for all participants. Given the relatively low nominal difficulty of the task, accuracy was, as expected, high and was similar for both groups. The overall accuracy for control participants was 97.4%, and the overall accuracy for immobilized participants was 96.4%. This information has been added to the Results section (lines 240-241).

Discussion

Thank you for raising this interesting point and recent finding. It is difficult to compare studies utilizing immobilization on the scale of hours to studies utilizing immobilization on the scale of days to weeks because an immobilization duration of a week has the potential to elicit larger effects on corticospinal excitability (Clark et al., 2010; Lundbye-Jensen et al., 2008) and even cortical thickness (Langer et al., 2012) that have not been previously shown in shorter (6hr) durations of immobilization. Although we did not observe a significant effect of immobilization on SRTT performance with the non-immobilized hand, we did observe small effects in the hypothesized direct, thus, we would predict a similar finding of increased non-immobilized performance on this task with longer periods of immobilization. This point has been added to the Discussion section (Section 4.3, lines 400-412).

Although we did observe small effects in the hypothesized direction, the degree of inter-individual variability in performance change during training detect potential between-group differences in performance within the training period. This information has also been added to the Discussion section (lines 488-493).

Minor

Why the TMS requirements had to be respected? In this study, TMS was not employed.

Thank you for identifying this oversight. The TMS inclusion criterion was part of a separate experiment, and it has been removed from this document for clarity.

Why did the authors choose a six-hour immobilization?

Previous literature has shown TMS markers of cortical plasticity as soon as three hours after the onset of immobilization (Karita et al., 2017). Six hours of immobilization was chosen in order for task training to occur after synaptic plasticity has been induced. Also, six hours compared to 8-12 hours was chosen to reduce participant burden and move towards clinical translation if it were to be used as a potential plasticity-inducing intervention. We have added discussion of this point in the Discussion section.

The name of the figures is wrong (figure 2 is called 3, and vice versa).

Thank you for identifying this error. We have corrected the figure titles.

I did not find in the text the link with the raw data.

We have added the link to the raw data within the text of the revised manuscript (line 143-144). The link can also be found here: https://osf.io/wazdy/?view_only=cfed7648613f45589c86d73582f93ce7

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PLoS One. doi: 10.1371/journal.pone.0276060.r003

Decision Letter 1

Benjamin A Philip

18 Apr 2022

PONE-D-21-19679R1Effects of short-term arm immobilization on motor skill acquisitionPLOS ONE

Dear Dr. Borich,

This manuscript is nearly ready for publication, but please address Reviewer 2's minor comments #1-2.

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The authors responded point-by-point to all comments.

I have only minor comments regarding their responses.

1) To test the homogeneity of variance, the authors should use Levene’s test, not Q-Q plots

2) When reporting the degree of freedom, they should report both the numerator and the denominator

3) The references have to be homogenized (e.g., l.68: A study by Opie, Evans (22)…)

Reviewer #2: The authors have substantially improved their manuscript and addressed all of my concerns. Thank you for taking into account my suggestions.

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PLoS One. 2022 Oct 14;17(10):e0276060. doi: 10.1371/journal.pone.0276060.r004

Author response to Decision Letter 1

1 Jun 2022

Response to Reviewer Comments:

1) To test the homogeneity of variance, the authors should use Levene’s test, not Q-Q plots

Thank you for this suggestion. We performed Levene’s test to assess homogeneity for each outcome measure across timepoints for both left and right hand data. The variances were not significantly different for any of the outcome measures. This information has been added to the manuscript.

2) When reporting the degree of freedom, they should report both the numerator and the denominator

Thank you for pointing this issue out. The numerator and denominator of the degrees of freedom (DFn, DFd) have been added to the tables.

3) The references have to be homogenized (e.g., l.68: A study by Opie, Evans (22)…)

The references have been edited throughout to improve consistency in formatting.

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PLoS One. doi: 10.1371/journal.pone.0276060.r005

Decision Letter 2

Benjamin A Philip

17 Jun 2022

PONE-D-21-19679R2Effects of short-term arm immobilization on motor skill acquisitionPLOS ONE

Dear Dr. Borich,

This manuscript is nearly ready for acceptance, but please address the remaining minor concerns by Reviewer 1.

Please submit your revised manuscript by Aug 01 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLoS One. 2022 Oct 14;17(10):e0276060. doi: 10.1371/journal.pone.0276060.r006

Author response to Decision Letter 2

2 Sep 2022

1) To test the homogeneity of variance, the authors should use Levene’s test, not Q-Q plots

2) When reporting the degree of freedom, they should report both the numerator and the denominator

Thank you for pointing this issue out. The numerator and denominator of the degrees of freedom (DFn, DFd) have been added to the tables.

3) The references have to be homogenized (e.g., l.68: A study by Opie, Evans (22)…)

The references have been edited throughout to improve consistency in formatting.

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PLoS One. doi: 10.1371/journal.pone.0276060.r007

Decision Letter 3

François Tremblay

28 Sep 2022

Effects of short-term arm immobilization on motor skill acquisition

PONE-D-21-19679R3

Dear Dr. Borich,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #2: (No Response)

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Reviewer #2: (No Response)

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Reviewer #2: (No Response)

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PLoS One. doi: 10.1371/journal.pone.0276060.r008

Acceptance letter

François Tremblay

5 Oct 2022

PONE-D-21-19679R3

Effects of short-term arm immobilization on motor skill acquisition

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

Raw behavioral data are available from the Open Science Framework database (link to view: https://osf.io/wazdy/?view_only=cfed7648613f45589c86d73582f93ce7).

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PERMALINK

Effects of short-term arm immobilization on motor skill acquisition

Erin M King

Lauren L Edwards

Michael R Borich

Roles

Abstract

1. Introduction

2. Materials and methods

2.1 Study participants

2.2 Motor task paradigm

Fig 1. Modified Serial Reaction Time Task (SRTT).

2.3 Arm immobilization

2.4 Motor skill acquisition paradigm

2.5 Behavioral outcome measures

2.5.1 Assessment of general motor performance

2.5.2 Assessment of sequence-specific skill

2.5.3 Task performance of the non-immobilized hand

3. Results

Fig 2. Immobilized participants complied with the immobilization procedure.

Table 1. Activity counts ANOVA results.

3.1 Assessment of general motor performance

Fig 3. Normalized SRTT response time across performance assessment timepoints.

Fig 4. General motor performance increased with task exposure in both groups.

3.2 Assessment of sequence-specific skill

Fig 5. Sequence-specific skill did not change after training and there was not evidence of an effect of immobilization.

Table 2. SRTT ANOVA results.

3.3 Task performance of the non-immobilized hand

Fig 6. Immobilization did not significantly influence performance of the right, non-immobilized hand.

Table 3. Nontarget hand motor performance.

4. Discussion

4.1 Lack of an effect of immobilization on motor performance on an individuated finger sequencing task

4.2 Acquisition of sequential, individuated finger movements was not preferentially enhanced after immobilization

4.3 Immobilization of the non-dominant limb did not influence dominant limb motor performance

4.4 Task characteristics may influence the effects of immobilization

4.5 Skill improvements across groups were not sequence-specific

4.6 Study limitations

5. Conclusions

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Benjamin A Philip

Roles

Author response to Decision Letter 0

Decision Letter 1

Benjamin A Philip

Roles

Author response to Decision Letter 1

Decision Letter 2

Benjamin A Philip

Roles

Author response to Decision Letter 2

Decision Letter 3

François Tremblay

Roles

Acceptance letter

François Tremblay

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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