Quadriceps Muscle Morphology Is an Important Determinant of Maximal Isometric and Crank Torques of Cyclists

of this study was to determine if quadriceps morphology [muscle volume (MV); cross-sectional area (CSA)], vastus lateralis (VL) muscle architecture, and muscle quality [echo intensity (ECHO)] can explain differences in knee extensor maximal voluntary isometric contraction (MVIC), crank torque (CT) and time-to-exhaustion (TTE) in trained cyclists. Twenty male competitive cyclists performed a maximal incremental ramp to determine their maximal power output (PO MAX). Muscle morphology (MV; CSA), muscle architecture of VL and muscle quality (ECHO) of both quadriceps muscles were assessed. Subsequently, cyclists performed three MVICs of both knee extensor muscles and nally performed a TTE test at PO MAXwith CT measurement during TTE. Stepwise multiple regression results revealed right quadriceps MV

maximal incremental ramp to determine their maximal power output (PO MAX). Muscle morphology (MV; CSA), muscle architecture of VL and muscle quality (ECHO) of both quadriceps muscles were assessed. Subsequently, cyclists performed three MVICs of both knee extensor muscles and nally performed a TTE test at PO MAXwith CT measurement during TTE. Stepwise multiple regression results revealed right quadriceps MV determined right MVIC (31%) and CT (33%). Left MV determined CT (24%); and left VL fascicle length (VL-FL) determined MVIC (64%). However, quadriceps morphological variables do not explain differences in TTE. No signi cant differences were observed between left and right quadriceps muscle morphology (p> 0.05). The ndings emphasize that quadriceps MV is an important determinant of knee extensor MVIC and CT but does not explain differences in TTE at PO MAX. Furthermore, quadriceps morphological variables were similar between the left and right quadriceps in competitive cyclists. Keywords: cyclists; performance predictors; maximal knee extensor torque; crank torque; quadriceps muscle properties 1. Introduction Endurance cycling performance is determined by physiological factors, such as maxi- mal oxygen uptake (VO2MAX), physiological transition thresholds, and metabolic ef ciency (e.g., cycling economy, gross ef ciency) [1–3]. VO2MAXis probably the most tested de- terminant for cycling performance [4,5]. Moreover, neuromuscular parameters have also been used to determine cycling time-to-exhaustion (TTE) and performance [6]. Miller and Manfredi [7] showed that physiological (i.e., anaerobic threshold) and anthropometric (i.e., thigh + calf/arm + chest) parameters are important performance determinants during a cycling time-trial (TT). Cycling endurance performance during TT is also explained by oxygen consumption (VO2), muscular hemoglobin concentration, and skeletal muscle oxygenation [8,9]. In addition, Lanferdini et al. [10] showed that the mechanical resultant pedaling force is a determinant of cycling submaximal performance as measured by the Sports2023,11, 22.

Sports2023,11, 22 2 of 14 power output (PO). Additionally, other studies have found moderate or strong correlations between physiological [11–13], or neuromuscular [14] variables with cycling endurance TT performance. Furthermore, a recent investigation showed that quadriceps muscle volume (MV) and vastus lateralis (VL) pennation angle (PA) determined 76% and 11% of the variance in peak power output (PPO) during sprint tests, respectively [15]. Therefore, in elite cyclists, VL-MV, in combination with the percentage of type-II muscle bers, explained 65% of variance in PPO during a Wingate test [8]. Focusing speci cally on knee extensor function, MV of the quadriceps femoris seems to be the best predictor (explaining 60%) of isometric knee extensor torque [16], and isokinetic PO during knee extension (explaining ~80%) in healthy subjects [17]. It has been suggested that MV is a determinant of maximal voluntary isometric contraction (MVIC) or crank torque (CT) in cyclists, but this remains to be determined. In addition, previous studies found a negative correlation between the echo inten- sity (ECHO; lower values mean better muscle quality) of rectus femoris (RF) and knee extensors' torque [18]. Similar ndings of a negative correlation between ECHO from gastrocnemius lateralis and soleus muscles and a positive correlation between MV of the triceps surae muscles [19] and the triceps surae's torque [19] have been reported in the literature. These results demonstrate that the lower the ECHO is (better muscle quality), the greater the torque production capacity of the assessed muscles [18,19]. However, the sample of both studies was composed of healthy subjects (men and women), non-cyclists or theelderly [16–19] . Furthermore, Song et al. [20] showed a moderate non-signi cant relationship between quadriceps ECHO (RF and vastus intermedius—VI) with maximum knee extensor strength. These results demonstrate a contradiction between ECHO and the ability to produce muscle strength. Moreover, no study was found relating ECHO to force production capacity during crank cycle. Although some studies have veri ed a relationship between muscle morphology (e.g., physiological cross-sectional area—PCSA), muscle architecture (fascicle length—FL) and skeletal muscle respiration with cycling aerobic performance [2,8,9,21], to date, no evidence has been found using different

between ECHO and the ability to produce muscle strength. Moreover, no study was found relating ECHO to force production capacity during crank cycle. Although some studies have veri ed a relationship between muscle morphology (e.g., physiological cross-sectional area—PCSA), muscle architecture (fascicle length—FL) and skeletal muscle respiration with cycling aerobic performance [2,8,9,21], to date, no evidence has been found using different variables of quadriceps femoris muscle morphology [i.e., MV, cross-sectional area (CSA)], muscle quality (i.e., ECHO), and VL muscle architecture to determine MVIC and CT during TTE in cyclists, nor have we found studies assessing a possible relationship between quadriceps muscle morphology, muscle quality and muscle architecture and TTE performance in cyclists. Therefore, the objective of this study was to determine if knee extensor MV, CSA, ECHO and VL muscle architecture were able to determine MVIC, CT and TTE performance in cyclists. If indeed cycling performance is somehow determined by quadriceps morphology, coaches and cyclists may decide whether to allocate time for training-speci c strength exercises aimed at quadriceps muscle hypertrophy during their regular endurance cycling training. 2. Materials and Methods 2.1. Experimental Approach We carried out a cross-sectional study to understand if quadriceps morphology and quality, and VL muscle architecture, are determinants of maximal isometric knee extensor torque and of crank torque during TTE performance. Each cyclist visited the laboratory on two occasions (Figure). During the rst visit, anthropometric data were assessed, and cyclists performed a maximal incremental test and familiarization to maximal TTE. After a week, athletes returned for the second visit. Muscle morphology (estimated MV; CSA), muscle quality (ECHO) and VL muscle architecture of both the left and right quadriceps' muscles were assessed. After that, cyclists performed three knee extensor MVICs with both lower limbs. Finally, cyclists performed a maximal TTE at maximal power output (POMAX) with measured CT. This study was conducted according to the Declaration of Helsinki, and all procedures were approved by the local Institutional Research Ethics Committee (project number 708.362). All cyclists were informed of the bene ts and risks of the investigation

a maximal TTE at maximal power output (POMAX) with measured CT. This study was conducted according to the Declaration of Helsinki, and all procedures were approved by the local Institutional Research Ethics Committee (project number 708.362). All cyclists were informed of the bene ts and risks of the investigation

Sports2023,11, 22 3 of 14 prior to signing an institutionally-approved informed consent document to participate in the study. Before each visit, subjects were instructed to avoid strenuous exercise and alcohol consumption within the last 48 h and to consume no caffeine or food during the nal 3 h before each test. The athletes participating in the present study had5.8 6.6 years of regular training/competition and no history of lower limb muscle-skeletal injuries. Exclusion criteria included chronic disease, smoking, metabolic disorders, use of steroids in the last six months, chronic disease, physical disabilities, smoking, and use of antibiotic drugs in the previous week.Sports 2023, 11, x FOR PEER REVIEW 5 of 5 Committee (project number 708.362). All cyclists were informed of the benefits and risks of the investigation prior to signing an institutionally-approved informed consent docu- ment to participate in the study. Before each visit, subjects were instructed to avoid stren- uous exercise and alcohol consumption within the last 48 h and to consume no caffeine or food during the final 3 h before each test. The athletes participating in the present study had 5.8 ± 6.6 years of regular training/competition and no history of lower limb muscle- skeletal injuries. Exclusion criteria included chronic disease, smoking, metabolic disor- ders, use of steroids in the last six months, chronic disease, physical disabilities, smoking, and use of antibiotic drugs in the previous week. Figure 1. Experimental design. Maximal power output (PO MAX). 2.2. Participants Twenty endurance-trained male cyclists participated in the study, having the follow- ing physical and physiological characteristics: Age 29.2 ± 6.6 years; b ody mass 77.1 ± 10.5 kg; height 179 ± 8 cm; PO MAX 377.6 ± 34.5 W; VO 2MAX 57.0 ± 7.7 mL∙kg∙min −1 ; training vol- ume 4.6 days and ~264 km/week; classified as performance level 3 (trained), according to De Pauw et al. [22]. Cyclists competed at the regional and national levels. 2.3. Procedures During the first session, anthropometric data were measured according to the Inter- national Society for the Advancement of Kinanthropometry [23]. After that, cyclists per- formed a warm-up with 150 W of

ume 4.6 days and ~264 km/week; classified as performance level 3 (trained), according to De Pauw et al. [22]. Cyclists competed at the regional and national levels. 2.3. Procedures During the first session, anthropometric data were measured according to the Inter- national Society for the Advancement of Kinanthropometry [23]. After that, cyclists per- formed a warm-up with 150 W of workload for 10 min . Cyclists were tested using a stand- ard road cycling bicycle (Giant TCR Advanced, Taichung, Taiwan ) with handlebars con- figuration and saddle position set to their anthropometrical characteristics. The bicycle was mounted on a stationary cycling trainer (CompuTrainer, ProLab 3D, Racermate Inc., Seattle, WA, USA) to determine PO MAX (in Watts). Before testing, tire pressure was cali- brated according to manufacturer instructions (~100 psi). Laboratory temperature (26- 28°C) and humidity (~50%) were controlled during all testing to minimize temperature effects on bicycle tire pressure and PO measurements [24]. Cyclists performed an incre- mental ramp test with 25 W increments every minute (~0.42 W/s) until exhaustion, using a custom-made script in cycling trainer software (CompuTrainer, CS 1.6, Racermate Inc, Seattle, WA, USA). Cadence was maintained close to 95 ± 5-rpm for all cyclists, using vis- ual feedback from the cycling trainer control unit. Exhaustion was defined by the follow- ing criteria: voluntary exhaustion or cadence dropping below 70 rpm. VO 2 was measured by an open- circuit indirect gas exchange system (CPX/D, Medical Graphics Corp., St. Louis, MO, USA) and VO 2MAX was defined as the greatest value obtained in the last stage of the incremental test, along with PO MAX. After incremental tests, cyclists pedaled for ~ 30 min at 50 W for recovery purposes and, finally, cyclists performed a familiarization with the TTE at PO MAX and a 95 ± 5- rpm of cadence. In the second session, quadriceps muscle morphology, muscle architecture and mus- cle quality were measured by the same investigator with extensive experience with ultra- sonography acquisition of muscles (~10 years). Quadriceps ultrasonography images were acquired using a B-mode Aloka ultrasound system (SSD 4000; ALOKA, Tokyo, Japan) with a 60- mm

PO MAX and a 95 ± 5- rpm of cadence. In the second session, quadriceps muscle morphology, muscle architecture and mus- cle quality were measured by the same investigator with extensive experience with ultra- sonography acquisition of muscles (~10 years). Quadriceps ultrasonography images were acquired using a B-mode Aloka ultrasound system (SSD 4000; ALOKA, Tokyo, Japan) with a 60- mm linear array transducer and 7.5 MHz. The ultrasonography probe was Figure 1.Experimental design. Maximal power output (PO MAX). 2.2. Participants Twenty endurance-trained male cyclists participated in the study, having the following physical and physiological characteristics: Age 29.2 6.6 years; body mass77.1 10.5 kg ; height 179 8 cm; POMAX377.6 34.5 W; VO2MAX57.0 7.7 mL kg min 1 ; training volume 4.6 days and ~264 km/week; classi ed as performance level 3 (trained), according to De Pauw et al. [22]. Cyclists competed at the regional and national levels. 2.3. Procedures During the rst session, anthropometric data were measured according to the Interna- tional Society for the Advancement of Kinanthropometry [23]. After that, cyclists performed a warm-up with 150 W of workload for 10 min. Cyclists were tested using a standard road cycling bicycle (Giant TCR Advanced, Taichung, Taiwan) with handlebars con guration and saddle position set to their anthropometrical characteristics. The bicycle was mounted on a stationary cycling trainer (CompuTrainer, ProLab 3D, Racermate Inc., Seattle, WA, USA) to determine POMAX(in Watts). Before testing, tire pressure was calibrated according to manufacturer instructions (~100 psi). Laboratory temperature (26–28 C) and humidity (~50%) were controlled during all testing to minimize temperature effects on bicycle tire pressure and PO measurements [24]. Cyclists performed an incremental ramp test with 25 W increments every minute (~0.42 W/s) until exhaustion, using a custom-made script in cycling trainer software (CompuTrainer, CS 1.6, Racermate Inc, Seattle, WA, USA). Cadence was maintained close to 95 5-rpm for all cyclists, using visual feedback from the cycling trainer control unit. Exhaustion was de ned by the following criteria: voluntary exhaustion or cadence dropping below 70 rpm. VO2was measured by an open-circuit indirect gas exchange system (CPX/D, Medical Graphics Corp., St. Louis, MO, USA)

(CompuTrainer, CS 1.6, Racermate Inc, Seattle, WA, USA). Cadence was maintained close to 95 5-rpm for all cyclists, using visual feedback from the cycling trainer control unit. Exhaustion was de ned by the following criteria: voluntary exhaustion or cadence dropping below 70 rpm. VO2was measured by an open-circuit indirect gas exchange system (CPX/D, Medical Graphics Corp., St. Louis, MO, USA) and VO2MAXwas de ned as the greatest value obtained in the last stage of the incremental test, along with POMAX. After incremental tests, cyclists pedaled for ~30 min at 50 W for recovery purposes and, nally, cyclists performed a familiarization with the TTE at POMAXand a 95 5-rpm of cadence. In the second session, quadriceps muscle morphology, muscle architecture and muscle quality were measured by the same investigator with extensive experience with ultra- sonography acquisition of muscles (~10 years). Quadriceps ultrasonography images were acquired using a B-mode Aloka ultrasound system (SSD 4000; ALOKA, Tokyo, Japan) with a 60-mm linear array transducer and 7.5 MHz. The ultrasonography probe was coated with a water-soluble gel to provide acoustic contact and was positioned on the skin without depressing the dermal surface. All ultrasonography images were acquired at rest with

Sports2023,11, 22 4 of 14 the lower limbs fully extended, after subjects rested for 10 min in a supine position on a stretcher. Three transversal ultrasound images were obtained for each muscle [VL, VI and RF, as well as the quadriceps muscle thickness (MT)] from both quadriceps' muscles (right and left). The probe was placed transversally (50% of the distance between the greater trochanter and the lateral femur condyle) using xed settings on the ultrasound equipment (frequency: 7.5 MHz; depth: 8 cm; General Gain: 40 dB; Time Gain Compensation—TGC in neutral position and focal zone 1.0 cm). After that, three longitudinal ultrasound images were obtained of VL (right and left) from each cyclist. After that, three longitudinal ultra- sound images were obtained of VL (right and left) from each cyclist, with the probe placed longitudinally to the muscle at 50% of the distance between the greater trochanter and the lateral femur condyle. Femur length was measured using a metric berglass tape (Sanny, S¢o Bernardo do Campo, Brazil, with 1 mm precision) from the distance between the femur's greater trochanter and the articular cleft between the femur and tibia condyles [25]. Quadriceps MV was estimated from the MT measurement between RF's super cial aponeurosis and VI's deep aponeurosis using ImageJ 1.42q software (National Institute of Health, Bethesda, MD, USA). Quadriceps MV was estimated using the equation proposed by Miyatani et al. [25], where: Quadriceps MV = [(Quadriceps MT (RF + VI) 320.6) + (femur length 110.9) 4437.9]. All ultrasound images were analysed by the same investigator with extensive expe- rience using ultrasonography analysis with the ImageJ 1.42q software (National Institute of Health, Bethesda, MD, USA). A maximum region of interest captured by the ultra- sonography probe (60-mm) was determined in each muscle and used to determine the quadriceps CSA and ECHO [26,27]. CSA measurements may have been underestimated due to the CSA's size of the assessed muscles, which, in some cases, exceeded the image's area captured by the ultrasound probe (60-mm). Mean grayscale of ECHO value of each muscle was determined using a standard grayscale histogram function and expressed as a

muscle and used to determine the quadriceps CSA and ECHO [26,27]. CSA measurements may have been underestimated due to the CSA's size of the assessed muscles, which, in some cases, exceeded the image's area captured by the ultrasound probe (60-mm). Mean grayscale of ECHO value of each muscle was determined using a standard grayscale histogram function and expressed as a value between 0 (black) and 255 (white) in the same software. The mean of three ultrasound images was used to quantify quadriceps CSA (RF + VI + VL) and ECHO [(RF + VI + VL)/3]; Figure. In addition, the VL best fascicle (i.e., the fascicle that was fully visible from its insertion on the deep aponeurosis to the super cial aponeurosis, or to the ultrasound probe eld-of-view end) in each ultrasonography image was used for muscle architecture analysis. FL was considered the length of the fascicular path between super cial and deep aponeuroses. When the ends of the fascicles were outside the ultrasound image, FL was estimated from extrapolation, as recommended in a previous study [28]. PA was calculated as the angle between the muscle fascicle and the deep aponeurosis. MT was considered a straight line between the deep and super cial aponeurosis along each ultrasonography image (Figure). Mean values were obtained from three ultrasound images for each muscle to determine FL, PA and MT of VL. The error in estimating the entire FL using the linear model ranged from 2–7% [28] to 13% [29]. After that, athletes were asked to sit on a chair of an isokinetic dynamometer (Biodex System 3 Pro, 2000 Hz, Biodex Medical Systems, Shirley, NY, USA) to perform MVIC of the knee extensor muscles, which was evaluated at 70 of knee exion (0 = full knee extension). After xation of the subject on the dynamometer chair, a verbal encouragement was given by researchers in each MVIC so that cyclists performed maximal torque in all contractions. All participants performed three 5-sec MVICs, with a 2-min rest interval between contractions. MVICs were measured from both lower limbs, and the highest or peak MVIC from each limb was

knee extension). After xation of the subject on the dynamometer chair, a verbal encouragement was given by researchers in each MVIC so that cyclists performed maximal torque in all contractions. All participants performed three 5-sec MVICs, with a 2-min rest interval between contractions. MVICs were measured from both lower limbs, and the highest or peak MVIC from each limb was used for further analysis.

This study investigates the relationship between quadriceps morphology and performance in trained cyclists.