1. Introduction
Pronated feet (PF) means reduced medial longitudinal arch at weight-bearing conditions. PF prevalence rates ranged from 48% to 78% in youth [1] and about 2%–23% in adults [2]. 
Individuals with over-pronated feet (OPF) demonstrate higher amplitude of tibialis posterior, tibialis anterior, toe flexors, medial gastrocnemius, and gluteus medius activities and lower amplitude of evertor activities compared to healthy individuals [3, 4, 5]. Electromyography (EMG) is a method to record the electrical activity of muscles [6]. The frequency content of EMG has been associated with muscle fatigue [7]. The frequency shift is associated with alterations in conduction velocity and changes in the recruitment of the motor units [7]. The median frequency during an isometric contraction is also related to the onset of muscle fatigue [8]. Accordingly, previous studies [3, 4] recommended that muscular activities should be regarded during the rehabilitation of OPF individuals.
Different procedures have been used in the rehabilitation of OPF [e.g. foot orthoses [9, 10] taping [11, 12, 13], and motion control shoes [14, 15]. Jafarnezhadgero et al., demonstrated that long-term running on sand led to reduced foot pronation and an increment in medial gastrocnemius amplitudes along with improved frontal plane pelvic stability due to higher gluteus medius amplitude [16]. However, these authors [16] did not evaluate the frequency content of muscles in their study. Sand has the potential to use as a suitable surface for rehabilitation sessions [17]. This study was conducted to assess the effect of long-term sand training on the muscular frequency content during running in runners with over-pronated feet.

2. Materials and Methods
G*Power software, version 3.1.9.4 was used to estimate the sample size (P=0.05, type error II=0.20, statistical power=80%, effect size=0.80) [18]. As a result, at least 30 samples are required to achieve large group×time interactions. Sixty male recreational runners with OPF participated in this randomized controlled trial study (Figure 1).

The eligibility criteria include a navicular drop >10 mm [19] a foot posture index >10 [20], a right foot dominant, a rearfoot striker, and physically active individuals. The exclusion criteria include limb length discrepancies of >5 mm, muscle spasms, neuromuscular or orthopedic-related disorders, or a history of surgery. Finally, sixty recreational OPF runners were randomly allocated to the IG and CG groups (Table 1). 

Experimental procedures
At first, a warm-up protocol (5 minutes) was performed [21]. Then, all participants ran barefoot on an 18-m runway at a speed of 3.3±5% m/s [22]. Five running trials during both pre-test and post-test were used for further data analysis [23]. All data collection process was done by a nave individual. 

Experimental setup and data processing
A force plate (Bertec Corporation, Columbus, OH, USA) was used to record ground reaction force (GRF) data (sampling rate of 1000 Hz). The GRF data was filtered through a cut-off frequency of 20 Hz [24]. Stance phase is defined as the period between ground contact (vertical GRF >10 N) to toe-off (GRF <10 N). Eight pairs of Ag/AgCl surface electrodes were used to record the muscle activities of tibialis anterior (TA), gastrocnemius medialis (GasM), biceps femoris (BF), semitendinosus (ST), vastus lateralis (VL), vastus medialis (VM), rectus femoris (RF), and gluteus medius (Gluteus-M) of dominant [8]. The EMG data were sampled at 1000 Hz using an EMG system (Biometrics Ltd, Nine Mile Point Ind. Est., New port, UK). First, the skin was cleaned with alcohol [25]. The stance phase was divided into loading (0%‒20% stance), mid-stance (20%‒50% stance), and push-of (50%‒100% stance) phases [26, 27, 28]. EMG data were high-pass and low-pass filtered with a cut-off frequency of 10 and 500 Hz, respectively. The median frequency of EMG data was then extracted for further analysis.

Sand running training protocol 
The sand running training program includes continuous barefoot jogging, striding, bounding, galloping, and short sprints for three sessions per week for 8 weeks [21, 29]. Each session included a 5-minute warm-up and stretching session, a 50-minute main training duration, and a 5-minute warm-down session [21]. For bounding, a straight-leg bound exercise was performed for a distance of 30 m [16]. For galloping, one foot was located in front of the opposite foot. The front foot took a large step forward, while the second foot remained in place. The 25 m sprints started with the subjects in a forward lunge status [16]. The IG was assessed after 6 days of the final training session [30]. Participants in the CG did not perform additional exercises during the study period. 

Statistical analyses
The normal distribution of data was affirmed using the Shapiro-Wilk test. A separate 2 (time: Pre vs post-test)×2 (groups: CG vs IG) analysis of variance (ANOVA) with repeated measures was computed. Effect sizes were determined using eta-squared (η2 p) [31]. The significance level was P<0.05. Statistical analyses were performed using SPSS software, version 24.

3. Results
Demographic variables were similar in both groups (Table 1).
The results demonstrated a significant group×time interaction for gluteus-M frequency content at the loading phase (P=0.012; η2=0.406) (Table 2).

Post-hoc analysis demonstrated an increase in frequency content for gluteus-M in the CG and decreases in the IG at the post-test than the pre-test. 
The results demonstrated a significant group×time interaction for gluteus-M frequency content at the mid-stance phase (P<0.001; η2=0.670) (Table 3).

Post-hoc analysis demonstrated an increase in frequency content for gluteus-M in the CG (but not in the IG) at the post-test than the pre-test. 
The results demonstrated a significant group×time interaction for Gas-M frequency content at the push-off phase (P=0.049; η2=0.298) (Table 4).

Post-hoc analysis demonstrated an increase in Gas-M frequency content in the IG (but not in the CG) at the post-test than the pre-test. 

4. Discussion
This study was conducted to evaluate the effect of long-term exercises on the sand on the muscular frequency content during running in runners with over-pronated feet.
The results demonstrated an increase in frequency content for gluteus-M in the CG and a decrease in the IG at the post-test compared to the pre-test. Foot pronation leads to internal tibia rotation and an anterolateral pelvic tilt. This may then lead to increased strain on pelvic muscles and a rotation of the lumbar vertebra during walking [32, 33, 34]. Decrease this strain via active contraction of erector spinae, theoretically, can lead to muscular fatigue [35]. As mentioned in a previous study, the reduced amount of gluteus-M frequency content in the intervention group may be associated with reduced foot pronation after sand training [16]. 
The results showed an increase in frequency content for gluteus-M in the CG (but not in the IG) at the post-test compared to the pre-test. Gluteus-M is a major muscle in motion of the pelvic hip complex in the frontal plane. During walking, gluteus-M provides the majority of support in the mid-stance phase [36]. It also works with the gluteus maximus to maintain the pelvis in the frontal plane [36]. Consequently, gluteus-M has a major role in daily activities, such as climbing [37], and running [38]. Weight-bearing exercises have been shown to generate higher gluteus-M activity than non-weight-bearing exercises [39, 40]. However, our results showed no changes in the gluteus-M activity after sand training in IG.
The results showed an increase in Gas-M frequency content in the IG (but not in the CG) at the post-test compared to the pre-test at the push-off phase. According to the literature [41], the relationship between the fascicles and tendons is crucial. In gait, the elastic energy cannot be provided directly from the impact due to the low Achilles force [42]. In running and jumping, the Achilles force increases during the loading phase [42]. In the running task, the Gas-M fascicles contracted together with high muscle activation during the loading phases [43]. A higher Gas-M frequency content in IG after training may lead to better force generation at the push-off phase. 
This study has limitations that should be regarded. First, only male elite runners were used in this study. Therefore, our results cannot be applied to women. Second, we did not record kinematic data in this study. 

5. Conclusion
As mentioned in a previous study, the reduced amount of gluteus-M frequency content in the intervention group may be associated with reduced foot pronation after sand training. A higher Gas-M frequency content in the IG after training may lead to better force generation at the push-off phase.

Ethical Considerations
Compliance with ethical guidelines
The present study was approved by the Ethical Committee of Ardabil University of Medical Sciences (Code: R.ARUMS.REC.1397.091), and registered at Iranian Registry of Clinical Trials (No.: IRCT20191211045704N1). 


Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Conceptualization, Visualization methodology, software, validation, formal analysis, investigation, resources, supervision, project administration, funding acquisition and review & editing: Amirali Jafarnezhadgero; Data curation, writing original draft: Kosar Gadehri and Ehsan Fakhri Mirzanag. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors thank the subjects who volunteered to participate in this study.


References

1. Chen KC, Tung LC, Tung CH, Yeh CJ, Yang JF, Wang CH. An investigation of the factors affecting flatfoot in children with delayed motor development. Research in Developmental Disabilities. 2014; 35(3):639-45. [DOI:10.1016/j.ridd.2013.12.012] [PMID]
2. Dunn JE, Link CL, Felson DT, Crincoli MG, Keysor JJ, McKinlay JB. Prevalence of foot and ankle conditions in a multiethnic community sample of older adults. American Journal of Epidemiology. 2004; 159(5):491-8. [DOI:10.1093/aje/kwh071] [PMID]
3. Murley GS, Landorf KB, Menz HB, Bird AR. Effect of foot posture, foot orthoses and footwear on lower limb muscle activity during walking and running: A systematic review. Gait & Posture. 2009; 29(2):172-87. [DOI:10.1016/j.gaitpost.2008.08.015] [PMID]
4. Hunt AE, Smith RM. Mechanics and control of the flat versus normal foot during the stance phase of walking. Clinical Biomechanics. 2004; 19(4):391-7. [DOI:10.1016/j.clinbiomech.2003.12.010] [PMID]
5. Gray EG, Basmajian JV. Electromyography and cinematography of leg and foot (“normal” and flat) during walking. The Anatomical Record. 1968; 161(1):1-15. [DOI:10.1002/ar.1091610101] [PMID]
6. Farrokhi S, Pollard CD, Souza RB, Chen YJ, Reischl S, Powers CM. Trunk position influences the kinematics, kinetics, and muscle activity of the lead lower extremity during the forward lunge exercise. Journal of Orthopaedic & Sports Physical Therapy. 2008; 38(7):403-9. [DOI:10.2519/jospt.2008.2634] [PMID]
7. De Luca CJ, Gilmore LD, Kuznetsov M, Roy SH. Filtering the surface EMG signal: Movement artifact and baseline noise contamination. Journal of Biomechanics. 2010; 43(8):1573-9. [DOI:10.1016/j.jbiomech.2010.01.027] [PMID]
8. Klein AB, Snyder-Mackler L, Roy SH, DeLuca CJ. Comparison of spinal mobility and isometric trunk extensor forces with electromyographic spectral analysis in identifying low back pain. Physical Therapy. 1991; 71(6):445-54. [DOI:10.1093/ptj/71.6.445] [PMID]
9. Alavi-Mehr SM, Jafarnezhadgero A, Salari-Esker F, Zago M. Acute effect of foot orthoses on frequency domain of ground reaction forces in male children with flexible flatfeet during walking. The Foot. 2018; 37:77-84.  [DOI:10.1016/j.foot.2018.05.003] [PMID]
10. Jafarnezhadgero A, Madadi-Shad M, Alavi-Mehr SM, Granacher U. The long-term use of foot orthoses affects walking kinematics and kinetics of children with flexible flat feet: A randomized controlled trial. PloS One. 2018; 13(10):e0205187. [DOI:10.1371/journal.pone.0205187] [PMID]
11. Aguilar MB, Abián-Vicén J, Halstead J, Gijon-Nogueron G. Effectiveness of neuromuscular taping on pronated foot posture and walking plantar pressures in amateur runners. Journal of Science and Medicine in Sport. 2016; 19(4):348-53.  [DOI:10.1016/j.jsams.2015.04.004] [PMID]
12. Cheung RT, Chung RC, Ng GY. Efficacies of different external controls for excessive foot pronation: A meta-analysis. British Journal of Sports Medicine. 2011; 45(9):743-51. [DOI:10.1136/bjsm.2010.079780] [PMID]
13. Luque-Suarez A, Gijon-Nogueron G, Baron-Lopez FJ, Labajos-Manzanares MT, Hush J, Hancock MJ. Effects of kinesiotaping on foot posture in participants with pronated foot: a quasi-randomised, double-blind study. Physiotherapy. 2014; 100(1):36-40. [DOI:10.1016/j.physio.2013.04.005] [PMID]
14. Jafarnezhadgero A, Alavi-Mehr SM, Granacher U. Effects of anti-pronation shoes on lower limb kinematics and kinetics in female runners with pronated feet: The role of physical fatigue. PloS One. 2019; 14(5):e0216818.  [DOI:10.1371/journal.pone.0216818] [PMID] 
15. Jafarnezhadgero AA, Sorkhe E, Oliveira AS. Motion-control shoes help maintaining low loading rate levels during fatiguing running in pronated female runners. Gait & Posture. 2019; 73:65-70. [DOI:10.1016/j.gaitpost.2019.07.133] [PMID]
16. Jafarnezhadgero A, Fatollahi A, Sheykholeslami A, Dionisio VC, Akrami M. Long-term training on sand changes lower limb muscle activities during running in runners with over-pronated feet. BioMedical Engineering Online. 2021; 20(1):118. [DOI:10.1186/s12938-021-00955-8] [PMID]
17. Binnie MJ, Dawson B, Pinnington H, Landers G, Peeling P. Sand training: A review of current research and practical applications. Journal of Sports Sciences. 2014; 32(1):8-15. [DOI:10.1080/02640414.2013.805239] [PMID]
18. Jafarnezhadgero A, Fatollahi A, Amirzadeh N, Siahkouhian M, Granacher U. Ground reaction forces and muscle activity while walking on sand versus stable ground in individuals with pronated feet compared with healthy controls. PloS One. 2019; 14(9):e0223219. [DOI:10.1371/journal.pone.0223219] [PMID]
19. Farahpour N, Jafarnezhad A, Damavandi M, Bakhtiari A, Allard P. Gait ground reaction force characteristics of low back pain patients with pronated foot and able-bodied individuals with and without foot pronation. Journal of Biomechanics. 2016; 49(9):1705-10. [DOI:10.1016/j.jbiomech.2016.03.056] [PMID]
20. Redmond AC, Crosbie J, Ouvrier RA. Development and validation of a novel rating system for scoring standing foot posture: the Foot Posture Index. Clinical Biomechanics. 2006; 21(1):89-98.  [DOI:10.1016/j.clinbiomech.2005.08.002] [PMID]
21. Durai DBJ, Shaju M. Effect of sand running training on speed among school boys. International Journal of Physical Education, Sports and Health. 2019; 6:117-22. [Link]
22. Anbarian M, Esmaeili H. Effects of running-induced fatigue on plantar pressure distribution in novice runners with different foot types. Gait & Posture. 2016; 48:52-6. [DOI:10.1016/j.gaitpost.2016.04.029] [PMID]
23. Pinnington HC, Lloyd DG, Besier TF, Dawson B. Kinematic and electromyography analysis of submaximal differences running on a firm surface compared with soft, dry sand. European Journal of Applied Physiology. 2005; 94(3):242-53. [DOI:10.1007/s00421-005-1323-6] [PMID]
24. Jafarnezhadgero AA, Shad MM, Majlesi M, Granacher U. A comparison of running kinetics in children with and without genu varus: A cross sectional study. PloS One. 2017; 12(9):e0185057.  [DOI:10.1371/journal.pone.0185057] [PMID]
25. Farahpour N, Jafarnezhadgero A, Allard P, Majlesi M. Muscle activity and kinetics of lower limbs during walking in pronated feet individuals with and without low back pain. Journal of Electromyography and Kinesiology. 2018; 39:35-41. [DOI:10.1016/j.jelekin.2018.01.006] [PMID]
26. Whittle MW. Gait analysis: An introduction. Amsterdam: Elsevier Science; 2014. [Link]
27. Dugan SA, Bhat KP. Biomechanics and analysis of running gait. Physical Medicine and Rehabilitation Clinics of North America. 2005; 16(3):603-21. [DOI:10.1016/j.pmr.2005.02.007] [PMID]
28. Shorter AL, Rouse EJ. Ankle mechanical impedance during the stance phase of running. IEEE Transactions on Biomedical Engineering. 2020; 67(6):1595-603. [DOI:10.1109/TBME.2019.2940927] [PMID]
29. Binnie MJ, Dawson B, Arnot MA, Pinnington H, Landers G, Peeling P. Effect of sand versus grass training surfaces during an 8-week pre-season conditioning programme in team sport athletes. Journal of Sports Sciences. 2014; 32(11):1001-12. [DOI:10.1080/02640414.2013.879333] [PMID]
30. Clarkson PM, Hubal MJ. Exercise-induced muscle damage in humans. American Journal of Physical Medicine & Rehabilitation. 2002; 81(11 Suppl):S52-69. [DOI:10.1097/00002060-200211001-00007] [PMID]
31. Cohen J. The effect size. Statistical Power Analysis for The Behavioral Sciences. 1988; 77-83. [Link]
32. Morra J. Electromyographic effects of foot orthotics on selected lower extremity muscles during running. Physical Therapy. 2000; 80(2):198-9. [Link]
33. Cowan SM, Bennell KL, Hodges PW, Crossley KM, McConnell J. Delayed onset of electromyographic activity of vastus medialis obliquus relative to vastus lateralis in subjects with patellofemoral pain syndrome. Archives of Physical Medicine and Rehabilitation. 2001; 82(2):183-9. [DOI:10.1053/apmr.2001.19022] [PMID]
34. Gupta A. Analyses of myo-electrical silence of erectors spinae. Journal of Biomechanics. 2001; 34(4):491-6. [DOI:10.1016/S0021-9290(00)00213-X] [PMID]
35. Bird AR, Bendrups AP, Payne CB. The effect of foot wedging on electromyographic activity in the erector spinae and gluteus medius muscles during walking. Gait & Posture. 2003; 18(2):81-91. [DOI:10.1016/S0966-6362(02)00199-6] [PMID]
36. Anderson FC, Pandy MG. Individual muscle contributions to support in normal walking. Gait & Posture. 2003; 17(2):159-69. [DOI:10.1016/S0966-6362(02)00073-5] [PMID]
37. Lyons K, Perry J, Gronley JK, Barnes L, Antonelli D. Timing and relative intensity of hip extensor and abductor muscle action during level and stair ambulation: An EMG study. Physical Therapy. 1983; 63(10):1597-605. [DOI:10.1093/ptj/63.10.1597] [PMID]
38. Chumanov ES, Wall-Scheffler C, Heiderscheit BC. Gender differences in walking and running on level and inclined surfaces. Clinical Biomechanics. 2008; 23(10):1260-8. [DOI:10.1016/j.clinbiomech.2008.07.011] [PMID]
39. Bolgla LA, Uhl TL. Electromyographic analysis of hip rehabilitation exercises in a group of healthy subjects. Journal of Orthopaedic & Sports Physical Therapy. 2005; 35(8):487-94. [DOI:10.2519/jospt.2005.35.8.487] [PMID]
40. Bauer A, Webright W, Arnold B, Schmitz R, Gansneder B. Comparison of weight bearing and non-weight bearing gluteus medius EMG during an isometric hip abduction. JAT. 1999; 34:S58.
41. Fukunaga T, Kubo K, Kawakami Y, Fukashiro S, Kanehisa H, Maganaris CN. In vivo behaviour of human muscle tendon during walking. Proceedings of the Royal Society of London Series B: Biological Sciences. 2001; 268(1464):229-33. [DOI:10.1098/rspb.2000.1361] [PMID] [PMCID]
42. Ishikawa M, Komi PV, Grey MJ, Lepola V, Bruggemann GP. Muscle-tendon interaction and elastic energy usage in human walking. Journal of Applied Physiology. 2005; 99(2):603-8. [DOI:10.1152/japplphysiol.00189.2005] [PMID]
43. French HP, Dunleavy M, Cusack T. Activation levels of gluteus medius during therapeutic exercise as measured with electromyography: A structured review. Physical Therapy Reviews. 2010; 15(2):92-105. [DOI:10.1179/174328810X12719009060380]