Ginger Root Extract Increases Mitochondrial Fission and Mitophagy in Diabetes Mellitus Rats

Diabetes (DM) is accompanied by mitochondrial dysfunction (i.e., mitochondria fission/fusion and mitophagy) in which result in an accumulation of damaged mitochondria and further impaired insulin resistance. Ginger root extract (GRE) has been shown to improve mitochondrial biogenesis and decreased respiratory coefficient in DM model, however, the effect of GRE on the basal mitochondria fission/fusion and mitophagy state is limited. PURPOSE: To determine the effect of GRE on mitochondria fission/fusion and mitophagy transcript abundance in rats with diabetes induced by high-fat diet (HFD) with streptozotocin (STZ). METHOD: Sprague-Dawley rats were randomly divided into 3 groups: standard diet (STD; n=11), HFD with 35 mg/kg of STZ (DM; n=11), and HFD+STZ with 0.75% w/w GRE (GRE; n=10). After 7 weeks, soleus samples were collected and analyzed for gene expression for fission/fusion (DRP, MFN) and mitophagy (PINK1, PARKIN, BECN1, LC3A, LC3B, P62). RESULT: A significant (p\u3c0.05) condition effect was found for PINK1, DRP, LC3A, LC3B, P62, and autophagic flux. For fission/fusion, GRE had significantly greater DRP (2.27±0.9-fold vs. 0.47±0.1-fold) than DM and no difference was found for MFN. For mitophagy, GRE had significantly greater PINK1 (1.59±0.55-fold vs. 0.31±0.06-fold), LC3A (1.81±0.65-fold vs. 0.13±0.02-fold), LC3B (2.71±0.92-fold vs. 0.66±0.25-fold), P62 (3.25±1.24-fold vs. 0.43±0.12-fold), and autophagy flux (4.5±1.06-fold vs. 2.41±0.36-fold) than DM and greater LC3B (2.71±0.92-fold vs. 1±0.06-fold), P62 (3.25±1.24-fold vs. 1±0.21-fold), and autophagic flux (4.5±1.06-fold vs. 1±0.26-fold) than STD. No difference was found for PARKIN and BECN1. CONCLUSION: In DM rats, GRE increased basal expression of mitochondria fission, degradation tag (PINK1), and autophagolysosome (LC3A, LC3B, P62, autophagic flux) markers, suggesting a potential increased in mitochondrial fission and mitophagy capacity

Curcumin Mitigates Neuropathic-Induce Muscle Atrophy by Suppressing CaMK2/NF-kB Pathway

Neuropathy can induce inflammation that results in muscle atrophy. Curcumin has been shown to exert an anti-inflammatory effect, preserving muscle mass in diabetic rats. However, the mechanism of curcumin to preserve muscle mass in neuropathy is not known. PURPOSE: To examine the effect of curcumin on the intramuscular inflammation and muscle cross-sectional area (CSA) in a neuropathy rodent model. METHODS: Twelve rats were randomly assigned to three groups: sham (CON), spinal nerve ligation (SNL), and SNL+100curcumin/kg BW (100CUR). The right (R) lumbosacral section (i.e., L5/L6) of the spinal cord was ligated (SNL and 100CUR) or sham surgery (CON) was performed, whereas the contralateral side (left: L) was served as their own control. Rats were fed with a control diet without (i.e., CON and SNL) or with (i.e., 100CUR) curcumin supplementation for 4 weeks. Plantaris (left and right) and tibialis anterior (TA; right) muscles were collected. TA was stained for dystrophin to measure CSA. Left and right plantaris were analyzed for protein content for AChR, CaMK2, CaMK2Thr286, CaMK2Thr286/CaMK2, NF-κB, NF-κBSer536, NF-κBSer536/NF-κB, IL-1β, and GAPDH. Each protein was normalized to GAPDH then to the CON. RESULTS: A significant (p ≤ 0.05) group effect was observed for TA CSA and a group x leg interaction effect was observed for CaMK2Thr286/CaMK2, NF-κBSer536, IL-1β protein content. For muscle CSA, CON (9027.33 ± 603.39 μm2) and 100CUR (8853.68 ± 696.73 μm2) were larger than SNL (4771.01 ± 539.69 μm2). No difference was observed in CSA between CON and 100CUR. Additionally, when compared between left and right plantaris muscles, only SNL had greater CaMK2Thr286/CaMK2 (R: 2.63 ± 1.87 vs. L: 1.56 ± 1.65), NF-κBSer536 (R: 1.85 ± 0.83 vs. L: 0.55 ± 0.33), and IL-1β (R: 2.11 ± 1.32 vs. L: 0.65 ± 0.29) protein content in the right than the left leg, whereas, no difference was observed for left leg among groups. For NF-κBSer536, SNL (1.85 ± 0.83) was greater than 100CUR (0.91 ± 0.52) in the right leg. No significant differences were observed for AChR, CaMK2, CaMK2Thr286, and NF-κBSer536/NF-κB. CONCLUSION: In a neuropathic model, muscle atrophy was observed with concomitant increase in CaMK2/NF-κB/IL-1β activation in the ipsilateral plantaris. Curcumin supplementation appears to mitigate this inflammatory response and muscle mass loss

Geranylgeraniol Supplementation Mitigates Muscle Atrophy with Mitochondrial Quality Improvement in Diabetic Rats

With diabetes, skeletal muscle mitochondrial quality control (mitochondrial fusion, fission & macro-autophagy) is impaired. Geranylgeraniol (GG) is shown to have a protective effect on preventing mitochondrial damage and muscle health; however, the effect of GG on a diabetic model is not known. PURPOSE: To determine the effect of GG on mitochondrial quality control and muscle cross-sectional area (CSA) in diabetic rats. METHODS: Thirty-five Sprague-Dawley rats were divided into three diet groups: control diet (CON), high-fat diet with 35 mg/kg body weight of streptozotocin (HFD), and HFD with 800 mg/kg body weight of GG (GG). Due to the limited sample, a total of 21 (CON: n = 7; HFD: n = 7; GG: n = 7) rats’ muscle samples were used for this report. The soleus muscles were harvested after 7-weeks of feeding and were analyzed for OPA1, MFN2, DRP1, pDRP, PINK1, Parkin, LC3A, and LC3B protein content using western blot analysis. Muscle CSA were assessed using Image J. RESULTS: A significant (p \u3c 0.05) condition effect was observed for MFN2, DRP, LC3A, and LC3B protein contents and muscle CSA. For mitochondrial fusion, GG (0.21 ± 0.08) had lower MFN2 than CON (0.43 ± 0.04; p = 0.007) and HFD (0.65 ± 0.08; p = 0.010). For mitochondrial fission, GG (0.26 ± 0.07) had lower DRP than HFD (0.59 ± 0.07; p = 0.019). For macro-autophagy, GG (1.08 ± 0.28) had lower LC3A than CON (2.81 ± 0.55; p = 0.028) and HFD (3.99 ± 0.57; p = 0.010); whereas GG (0.63 ± 0.21) had lower LC3B than HFD (1.93 ± 0.24; p = 0.012). No significant differences were observed for OPA1, pDRP, PINK1, Parkin, and LC3B/A. For muscle size, CON (10,092.88 ± 104.67µm2) had larger CSA than GG (7284.69 ± 70.91µm2, p = 0.001) and HFD (5615.59 ± 59.97µm2; p = 0.001), whereas GG (7284.69 ± 70.91µm2) had larger CSA than HFD (5615.59 ± 59.97µm2; p = 0.001). CONCLUSION: GG supplementation could prevent mitochondrial fragmentation (reduction in DRP), thus, potentially resulting in a decreased demand for mitochondrial fusion (reduction in MFN2). In addition, a greater rate of autophagosome degradation than formation (reduction in LC3A and LC3B) was observed (indicative of an increase in macro-autophagy). Improvement in mitochondrial quality could potentially contribute to attenuating the reduction of muscle size in diabetic rats with GG supplementation

Muscular Torque Output During Neuromuscular Electrical Stimulation Following a 4-week Training Intervention in Older Adults

Neuromuscular electrical stimulation (NMES) can be used to induce muscle torque by generating involuntary muscle contractions. If a greater muscular torque and torque maintenance could be induced by NMES training bouts, it may lead to improvements in electrically induced muscular endurance. However, little is known regarding torque output during NMES pre-post training. PURPOSE: The purpose of this study was to determine if a 4-week NMES training intervention would alter involuntary muscular torque output during the NMES protocol in older, healthy adults. METHODS: Eleven older adults (68.7 ± 2.1 years) completed 12 (Day 1 – Day 12), 40-min NMES training sessions of the quadriceps muscles three times a week, over 4-weeks, with the stimulation frequency set at 60 Hz. Maximal voluntary contractions (MVC) were measured pre-training and mid-training. NMES was delivered through stimulation electrodes placed on the quadriceps muscles and torque output was recorded during the training sessions. Stimulation intensity was set to generate muscular torque output to meet a target representing 15% MVC and was adjusted every 5 minutes to achieve target torque. During each training session, 96 total contractions were generated during the NMES protocol. For Day 1 and Day 12, mean torque, peak torque, and torque time integral (TTI) were measured for each contraction and were then normalized to the pre-training MVC for Day 1 and mid-training MVC for Day 12. The overall mean of the 96 contractions was then calculated for each torque parameter. Sum of TTI (STTI) was calculated by summing the normalized TTI for all contractions. The average stimulation intensity was recorded, and the mean was calculated for each day. Paired sample t-tests were used to test for differences between Days (Day 1 and Day 12) for torque parameters and stimulation intensity. Statistical significance was set at p ≤ 0.05. RESULTS: TTI (Day 1: 90.5 ± 6.1% MVC vs Day 12: 75.9 ± 9.4% MVC; p = 0.036) and STTI (Day 1: 8,686.4 ± 582.0% MVC vs Day 12: 7,2801.0 ± 903.8% MVC; p = 0.036) were lower on Day 12 compared to Day 1. Additionally, there was a trend toward lower mean torque after training (Day 1: 8.7 ± 0.5% MVC vs Day 12: 7.3 ± 0.9% MVC; p = 0.055). Peak torque was not different between days (Day 1: 12.9 ± 0.6% MVC vs Day 12: 13.0 ± 0.7% MVC; p = 0.859). Stimulation intensity showed a trend toward higher stimulation intensity on Day 12 compared to Day 1 (Day 1: 13.3 ± 0.7 mA vs Day 12: 14.6 ± 1.0 mA, p = 0.10). CONCLUSION: Torque output during the NMES protocol was not improved with NMES training and demonstrated a decrease in some torque parameters. The inability of the muscle to produce similar torque output after training may be due to muscle accommodation to the NMES stimulation with repeated bouts. If the goal is to improve involuntary muscular endurance, allowing for more recovery between NMES sessions and use of a lower stimulation frequency may facilitate greater overall muscular torque output following NMES training

Geranylgeraniol Increases Autophagy and Mitophagy Gene Expression in Soleus of Rats with Diabetes

The autophagy and mitophagy (selective autophagy for mitochondria) processes are important in maintaining muscle homeostasis, e.g., removing damaged mitochondria. With diabetes, skeletal muscle autophagy decreases. Geranylgeraniol (GG) has been shown to reduce mitochondrial damage; however, the effect of GG on basal autophagy and mitophagy in diabetic rats is not known. PURPOSE: To determine the effect of GG on selective autophagy and mitophagy genes in rats with diabetes induced by a high-fat diet (HFD) with streptozotocin (STZ). METHODS: Sprague-Dawley rats were divided into three groups: normal diet (CON; n=11), HFD with 35 mg/kg body weight of STZ (HFD; n=9), and HFD/STZ with 800 mg/kg body weight of GG (GG; n=9). On the 7th week, soleus muscles were collected and analyzed for gene expression of LC3A, LC3B, P62, PINK1, PARKIN, DRP, and MFN. Gene data were normalized to CON. RESULTS: A significant (p \u3c 0.05) condition effect was found for autophagy (LC3A, LC3B, and P62) and mitophagy (PINK1, DRP, and MFN) gene expression. For autophagy, HFD (0.14 ± 0.03-fold) had significantly lower LC3A than CON (1.00 ± 0.22-fold), lower LC3B (0.67 ± 0.26-fold vs. 2.37 ± 0.72-fold) and P62 (0.44 ± 0.13-fold vs. 1.70 ± 0.35-fold) than GG. HFD trended to have lower LC3A than GG (0.14 ± 0.03-fold vs. 0.86 ± 0.26-fold; p = 0.066) while CON trended to have lower LC3B than GG (1.00 ± 0.07-fold vs. 2.37 ± 0.72-fold; p = 0.078). For mitophagy, HFD (0.32 ± 0.07-fold) and GG (0.51 ± 0.15-fold) had significantly lower PINK1 than CON (1.00 ± 0.13-fold). Further, HFD had lower MFN than GG (0.31 ± 0.08-fold vs. 1.46 ± 0.25-fold) and lower DRP than CON (0.48 ± 0.11-fold vs. 1.00 ± 0.15-fold). HFD trended to have lower PARKIN than GG (0.46 ± 0.08-fold vs. 1.38 ± 0.38-fold; p = 0.053) with no difference between GG and CON. CONCLUSION: In comparison to HFD, GG consumption improved the basal transcript abundance of the selective skeletal muscle autophagic and mitophagic genes, which could indicate an increased capacity to remove damaged mitochondria in diabetic rats

The Effect of Geranylgeraniol on Satellite Cells Myogenic State in Type 2 Diabetic Rats

Type 2 Diabetes (T2D) is associated with chronic inflammation, which can contribute to impaired satellite cells (SC) myogenic state that may result in muscle atrophy. Geranylgeraniol (GGOH) has shown to prevent muscle atrophy, reduce inflammatory markers, and increase SC content; however, the effect of GGOH on SC myogenic state in T2D rats is not known. PURPOSE: To examine the effects of GGOH on SC myogenic state and muscle cross-sectional area (CSA) in T2D rats. METHODS: 21 Sprague-Dawley rats were fed a control diet (CON; n=7), a high-fat diet with 35 mg/kg of streptozotocin (HFD; n=7), and HFD with 800mg/kg body weight of GGOH (GG; n=7). In the 8th week, the right soleus muscle was analyzed for protein expression for Pax7, MyoD, myostatin, and GAPDH, and protein content was normalized to GAPDH. The left soleus muscle was co-stained with Pax7, MyoD, and myostatin using immunohistochemistry and analyzed for muscle CSA. Counted SC were normalized to 100 fibers. RESULTS: A significant (p \u3c 0.05) condition effect was observed for MyoD and myostatin protein expression. For MyoD, HFD (1.41 ± 0.09 A.U.) was lower than CON (2.24 ± 0.21 A.U.) and GG (2.62 ± 0.43 A.U.). For myostatin, HFD (0.42 ± 0.06 A.U.) was lower than CON (0.91 ± 0.09 A.U.). Additionally, a significant condition effect was observed for the number of cells that presented Pax7+/MyoD- and Pax7+/myostatin+. For Pax7+/MyoD-, HFD (0.039 ± 0.004) and GG (0.035 ± 0.004) had lower cell counts than CON (0.064 ± 0.010). For Pax7+/myostatin+, HFD (0.034 ± 0.003) had lower cell counts than GG (0.065 ± 0.010) and CON (0.057 ± 0.004). A significant condition effect was observed for CSA where CON (7099.89 ± 187.33 μm2) was larger than HFD (4351.02 ± 127.46 μm2) and GG (5584.61 ± 208.01 μm2), while GG (5584.61 ± 208.01 μm2) was larger than HFD (4351.02 ± 127.46 μm2). CONCLUSION: GGOH supplementation to T2D rats mitigated muscle mass loss (increased MyoD expression with no change in MyoD+ SC). Despite no differences in SC myogenic state (proliferative and differentiation) among groups, GGOH appeared to mitigate the reduction in the quiescent SC pool (Pax7+/myostatin+) observed in HFD. Given the importance of quiescent SC pool on retaining myogenic potential, which is essential for muscle hypertrophy and regeneration, supplementing GGOH to T2D rats could improve muscle health

Sex Differences in The Accuracy of WUT (Weight, Urine Color, Thirst) Diagrams Assessing Hydration Status

The WUT (Weight, Urine Color, Thirst) Venn diagram is a practical method to assess hydration status using percent body mass loss (%BML), urine color (UCOL), and thirst perception (TP). However, sex differences and the accuracy of WUT diagrams between males and females has not yet been investigated. PURPOSE: To observe sex differences in the accuracy of WUT diagrams assessing hydration status. METHODS: 8 males [M] (age: 21 ± 3; mass: 76.3 ± 15.6 kg) and 5 females [F] (age: 22 ± 2; mass: 60.5 ± 13.6) visited the laboratory twice a day (morning (7:00am-9:00am) and afternoon (2:00pm-4:00pm)) for six days as free-living for the first three consecutive days and euhydrated (urine specific gravity (USG) \u3c 1.020) for the last three consecutive days. During each visit, TP, body mass (BM), USG, UOSM, UCOL, and plasma osmolality (POSM) were collected. Values of USG \u3e1.020, UOSM \u3e700, and POSM \u3e290 indicated dehydration status. TP \u3e5, UCOL \u3e5, and %BML \u3e1% values were used as dehydration thresholds for WUT scores. Total WUT score (0-3) was determined by the total amount of respective dehydration markers identified. One-way ANOVA was used to analyze differences in POSM, UOSM, and USG between the different WUT scores for both sexes. Receiver operating characteristics analysis was used to calculate sensitivity (SENS) and specificity (SPEC) identifying dehydration or euhydration with WUT scores. RESULTS: For POSM, WUT3 (M: 291 ± 5; F: 286 ± 0 mOsmol), WUT2 (289 ± 6; 286 ± 7), WUT1 (286 ± 5; 286 ± 6), and WUT0 (289 ± 5; 285 ± 7) were not different between sexes (p \u3e .05). For USG, WUT3 (1.022 ± .004; 1.020 ± .000), WUT2 (1.019 ± .008; 1.020 ± .007), WUT1 (1.015 ± .006; 1.010 ± .005), and WUT0 (1.010 ± .006; 1.008 ± .006) were not different between sexes (p \u3e .05). For UOSM, WUT3 (819 ± 147; 744 ± .000 mOsmol), WUT2 (679 ± 244; 788 ± 261), WUT1 (521 ± 266; 461 ± 212), and WUT0 (383 ± 212; 322 ± 203) were not different between sexes (p \u3e .05). For POSM, WUT2SPEC was higher in M (WUT2Mspec, .860) than F (WUT2Fsepc, .786) while WUT3, WUT1, and WUT0 were similar between sexes (WUT3Mspec, .965; WUT3Fspec, .976; WUT1Mspec, .526; WUT1Fspec, .380). For USG, WUT2SENS was higher in F (WUT2Fsens, .889) than M (WUT2Msens, .571) while WUT3, WUT1, and WUT0 were similar between sexes (WUT3Msens, .238; WUT3Fsens, .111; WUT1Msens, .905; WUT1Fsens, .889). For UOSM, SPEC and SENS were similar between sexes for each WUT score. CONCLUSION: There are no sex differences in POSM, USG, and UOSM between WUT0-WUT3. However, based on SPEC and SENS, WUT3 and WUT0 can accurately detect hydration status in both sexes. WUT2 might be used to detect hydration status only for females

Changes in Physical Function Following 4-Weeks of Neuromuscular Electrical Stimulation Training in Older Adults

Sarcopenia, the age-related loss of muscle mass and strength, can result in a decline in physical function. Neuromuscular electrical stimulation (NMES) has been shown to induce muscular adaptations that have the potential to translate to functional improvements; however, little is known regarding functional adaptations pre-post short-term NMES training, especially in older adults. PURPOSE: The aim of this study was to determine NMES-induced changes in lower extremity physical function following 4 weeks of an NMES training intervention of the quadriceps muscle in older adults. METHODS: Seventeen healthy, older adults (68.8 ± 1.8 years old) were divided into two groups: NMES (n = 12) and SHAM (n = 5). The NMES group underwent 12, 40-minute NMES training sessions to the quadriceps muscles on each leg 3x/week over 4 weeks, with the stimulation intensity adjusted every 5 minutes, as needed, to achieve a 15% target torque of each participant’s maximal voluntary contraction (MVC). The stimulation parameters consisted of a 60 Hz stimulation frequency and a duty cycle of 10s on and 15s off. The SHAM group was blinded and did not receive any treatment. The following functional assessments were measured before and after the 4-week training period: Timed Up and Go (TUG), 5x Sit-to-Stand (5XSTS), Stair Climb (SC), and 6-Minute Walk Test (6MWT). Repeated-measures ANOVAs were used to determine changes in TUG, 5XSTS, SC, and 6MWT assessments pre-post NMES training and data are reported as mean ± SE. Statistical significance was set at P \u3c 0.05. RESULTS: NMES training significantly improved TUG (NMES: 8.81 ± 0.54s vs. 7.67 ± 0.39s; P = 0.002; SHAM: 10.60 ± 2.41 vs. 10.93 ± 3.01s; P = 0.652; pre- and post-training, respectively) and SC (NMES: 4.03 ± 0.20s vs. 3.76 ± 0.16s; P = 0.023; SHAM: 6.53 ± 2.11 vs. 6.0 ± 1.78s; P = 0.215; pre- and post-training, respectively); however, 5XSTS (NMES: 9.70 ± 0.75 vs. 8.83 ± 0.72; P \u3e 0.05; SHAM: 14.34 ± 3.64 vs. 13.28 ± 3.89; P \u3e 0.05; pre- and post-training, respectively) and 6MWT (NMES: 610.10 ± 22.68 vs. 623.74 ± 14.73; P \u3e 0.05; SHAM: 533.43 ± 82.44 vs. 587.81 ± 80.52; P \u3e 0.05; pre- and post-training, respectively) did not change following the NMES intervention. CONCLUSION: Improvements in TUG and SC following 4 weeks of NMES training demonstrate augmented lower body physical function, suggesting that short-term NMES training programs may induce neuromuscular adaptations that contribute to these early improvements in physical function in older adults

The Effect of Morning Thirst on Afternoon Hydration Status

Thirst sensation is an important stimulus for drinking behavior; however, the effect of thirst sensation on later hydration status remains unclear. PURPOSE: To investigate the effects of morning thirst on afternoon hydration status. METHODS: Twelve men (mean ± standard deviation; age: 21 ± 2 years; mass: 81.0 ± 15.9 kg) and twelve women (age: 22 ± 3 years; mass: 68.8 ± 15.2 kg) visited the laboratory in the morning (first morning) and afternoon (2:00-4:00pm) for three consecutive days under a free-living condition. At each visit, participants provided a urine sample where urine indices were analyzed (urine specific gravity [USG], urine color [UCOL], urine osmolality [UOSMO]), and nude body mass was collected to calculate body mass loss (BML). Then, thirst was assessed with a Likert scale, and a blood sample was collected to analyze plasma osmolality (POSMO), hemoglobin, and hematocrit to calculate % plasma volume change (PV). Participants recorded food and fluid intake between the morning and afternoon visits to determine total water intake (TWI). Linear regression was used to predict thirst from the morning on hydration indices in the afternoon. Also, a stepwise linear regression predicted thirst in the afternoon from hydration indices in the morning. Pearson’s product moment correlation was used to calculate the relationship between TWI and hydration markers. RESULTS: Higher morning thirst significantly predicted lower UOSMO (r2=0.056, p=0.045), USG (r2=0.096, p=0.008), UCOL (r2=0.074, p=0.021), and higher thirst (r2=0.074, p=0.021) in the afternoon. However, thirst in the morning did not predict BML, PV, POSMO, and TWI in the afternoon (p\u3e0.05). Increased thirst and BML in the morning together significantly predicted higher thirst (r2=0.125, p=0.010) in the afternoon. Increased TWI was associated with lower UCOL (r=0.336, p=0.004) and BML (r=0.297, p=0.011) in the afternoon. However, TWI was not associated with any variables in the morning or the remaining variables in the afternoon, including USG and UOSMO (p\u3e0.05). CONCLUSION: Increased morning thirst impacts afternoon urine indices and thirst. However, morning thirst does not influence TWI between the morning and the afternoon, and TWI is not associated with morning hydration status. Therefore, afternoon hydration status might be impacted by morning thirst, although individuals might not consume fluid based on their morning thirst or hydration status

Neuromuscular Electrical Stimulation Effects on Skeletal Muscle Fatigue in Older Adults

Neuromuscular electrical stimulation (NMES) is often used as a rehabilitative modality and evidence has suggested that high frequencies of NMES may elicit increases in muscle strength. However, little is known regarding the effects of a high-frequency NMES intervention on voluntary skeletal muscle fatigue. PURPOSE: The aim of this study was to determine the effect of a 4-week high-frequency NMES intervention on voluntary muscular fatigue and changes in neuromuscular activation patterns of the quadriceps during voluntary fatiguing muscle contractions in older adults. METHODS: Seventeen healthy, older adults (68.8 ± 1.8 years old) participated in the study (NMES: n = 12; SHAM: n = 5). Each participant was seated on an isokinetic dynamometer, and a 40-min NMES treatment was applied to the quadriceps muscles of each leg 3x/week for 4 weeks with the stimulation frequency set at 60 Hz. Stimulation intensity was set to achieve 15% of knee extension maximal voluntary contraction (MVC). Those in the SHAM group underwent the same treatment procedures but did not receive the NMES treatment. All subjects performed maximal voluntary contractions (MVC) and an intermittent knee extension isometric submaximal voluntary fatigue task at 50% MVC until the fatigue criteria were met for pre-post testing. Surface electromyography (sEMG) of the vastus lateralis (VL) and vastus medialis (VM) muscles were recorded during the fatigue task to examine changes in muscle activation. EMG data were quantified for root mean square (RMS) EMG and reported as a percent rate of change over the duration of the fatigue task and median frequency (MF) is reported as the average MF during the fatigue task. Repeated measures ANOVAs were used to determine differences pre-post NMES for muscular endurance time, MVC and EMG measures. Statistical significance was set at p \u3c 0.05. RESULTS: MVC increased pre-post NMES in the NMES group (117.1 ± 8.7 Nm vs 127.6 ± 11.1 Nm, p = 0.049; pre- and post-training, respectively) with no change in SHAM (p = 0.96). Muscular endurance time did not change pre-post NMES (NMES: 159.3 ± 20.1s vs 141.9 ± 21.2s, p = 0.29; SHAM: 242.2 ± 43.3s vs 202.9 ± 23.3s, p = 0.13; pre- and post-training, respectively). RMS EMG rate of change did not change following NMES treatment (NMES: VL: 16.6 ± 3.6% vs 18.8 ± 10.4%, p = 0.84; VM: 11.4 ± 2.1% vs 19.6 ± 5.5%, p = 0.15; SHAM: VL: 7.8 ± 1.6% vs 7.1 ± 3.0%, p = 0.81; VM: 7.1 ± 3.3% vs 5.9 ± 2.2%, p = 0.55; pre- and post-training, respectively). Also, there was no difference in MF EMG with NMES training (NMES: VL: 77.6 ± 4.1 Hz vs 74.9 ± 3.6 Hz, p = 0.13; VM: 72.5 ± 2.4 Hz vs 72.6 ± 2.2 Hz, p = 0.97; SHAM: VL: 79.3 ± 3.4 Hz vs 80.2 ± 4.9 Hz, p = 0.85; VM: 76.9 ± 3.7 Hz vs 83.9 ± 5.1 Hz, p = 0.12; pre- and post-training, respectively). CONCLUSION: Treatment with high-frequency NMES did not improve muscle endurance or related EMG parameters. It is possible that NMES induced adaptations may be frequency-specific and that high-frequency NMES may not be efficacious when the goal is to improve skeletal muscle endurance