Anaerobic and Aerobic Metabolic Capacities Contributing to Yo-Yo Intermittent Recovery Level 2 Test Performance in Australian Rules Footballers

study aimed to identify the aerobic and anaerobic metabolic performance capaci- ties contributing to Yo-Yo Intermittent Recovery level 2 (Yo-Yo IR2) test performance. Nineteen recreational Australian footballers completed a Yo-Yo IR2 test, and on another day a treadmill peak oxygen uptake (VO 2peak) and maximal accumulated oxygen deficit test in a randomised counter- balanced order. The maximal accumulated oxygen deficit (MAOD) procedures included 5×5 min sub-maximal continuous runs at progressively higher speeds whilst VO 2was recorded; thereafter, speed was incrementally increased to elicit VO 2peak. After 35 min of rest, participants ran at a speed equivalent to 115% of VO 2peakuntil exhaustion, at which point expired air was collected to determine maximal accumulated oxygen deficit. Relationships between variables were assessed using Pearson’s correlation and partial correlations. Maximum aerobic speed, relative intensity, and VO 2peakwere significantly correlated with Yo-Yo IR2 performance. High Yo-Yo IR2 performers also had higher MAS, relative intensity, and VO 2peaklevels. However, when higher maximum aerobic speed, relative intensity, and VO 2peakwere controlled for each other and analysed independently, neither maxi- mal aerobic speed nor VO 2peakcorrelated with Yo-Yo IR2 performance. Yo-Yo IR2 performance is the result of a complex

VO 2peakwere significantly correlated with Yo-Yo IR2 performance. High Yo-Yo IR2 performers also had higher MAS, relative intensity, and VO 2peaklevels. However, when higher maximum aerobic speed, relative intensity, and VO 2peakwere controlled for each other and analysed independently, neither maxi- mal aerobic speed nor VO 2peakcorrelated with Yo-Yo IR2 performance. Yo-Yo IR2 performance is the result of a complex interaction between several variables. Training programs should primarily focus on improving VO 2peak,maximal aerobic speed, and relative intensity to optimize Yo-Yo IR2 test performance. Keywords:maximal aerobic speed; aerobic power; running economy; maximal accumulated oxygen deficit 1. Introduction Australian Rules football requires athletes to perform numerous high-intensity efforts with short recovery [1]. Like other team sports, Australian Rules football coaches and physical trainers perform several field tests to identify the physiological and metabolic capabilities of athletes to cope with the repeated high-intensity demands of a match [2–4]. The Yo-Yo Intermittent Recovery level 2 test (Yo-Yo IR2) is a field test that strongly correlates with the high-speed locomotion in Australian Rules football and soccer [5,6]. One of the strengths of the Yo-Yo IR2 test in predicting Australian Rules football and soccer repeated-sprint performance is that it challenges a variety of physiological systems that interact in a similar way to match play in many team sports [7,8]. Further, it has been suggested that tests such as the Yo-Yo could be included as part of multi-measurement criteria for player selection, especially when it is challenging to discriminate sport-specific fitness between players [9]. Likewise, selected state-level Australian Football League (AFL) women players have been observed to perform better in the Yo-Yo IR1 test compared to their non-selected counterparts [10]. Differences exist between the Yo-Yo IR1 and IR2, demonstrating that Yo-Yo variations may be discriminating factors in player selection. Sports2024,12, 236.

Sports2024,12, 236 2 of 8 Given the demonstrated links between Yo-Yo IR2 performance and team sport exercise intensity, as well as possible impacts on player selection, team sport training programs may attempt to measure the success of training interventions using this test (8). However, to improve Yo-Yo IR2 performance, knowledge of the relative influence of the aerobic and anaerobic metabolic capacities underlying its performance is essential to optimize a specific and periodized training regime. Aerobic metabolic capacity contributes substantially to Yo-Yo IR2 test performance as the test elicits maximum heart rate [11]. Yoshida and Watari [12] showed that distance runners with a high peak oxygen uptake (VO 2peak) can resynthesize phosphate creatine more quickly than runners with lower VO2. In theory, quicker phosphate creatine resynthesis should enhance repeated high-intensity efforts. However, Rampinini et al. [13] showed that VO 2peakonly moderately correlates with Yo-Yo IR2 (r = 0.47). Additionally, VO 2peakis not a strong discriminator of endurance sports performance in heterogenous samples, and other aerobic performance qualities such as maximal aerobic speed (MAS) and running economy can be more predictive of endurance performance [14]. Olsen et al. [15] recently showed that maximal aerobic speed correlates strongly with repeated-sprint ability in soccer players. The relationship of maximal aerobic speed and running economy to Yo-Yo IR2 performance has not been previously investigated. Yo-Yo IR2 test performance has large correlations with post-test blood lactate, H + accumulation, and rate of lactate accumulation [7]. Consequently, a high anaerobic capacity appears to be an important determinant of Yo-Yo IR2 performance, as maximal Yo-Yo IR2 performance requires a sustained contribution from anaerobic glycolysis. As the influence of aerobic metabolic performance measures on Yo-Yo IR2 performance is unclear and the relationship of anaerobic capacity to Yo-Yo IR2 performance has not been previously investigated, this study aims to determine how these metabolic qualities correlate with Yo-Yo IR2 performance. The hypothesis tested is that aerobic metabolic performance and anaerobic capacity measures are correlated with Yo-Yo IR2 performance. 2. Materials and Methods 2.1. Subjects Nineteen male regional team sport athletes (Australian Rules footballers) from various teams volunteered to participate in this

has not been previously investigated, this study aims to determine how these metabolic qualities correlate with Yo-Yo IR2 performance. The hypothesis tested is that aerobic metabolic performance and anaerobic capacity measures are correlated with Yo-Yo IR2 performance. 2. Materials and Methods 2.1. Subjects Nineteen male regional team sport athletes (Australian Rules footballers) from various teams volunteered to participate in this study in response to advertising flyers placed at football clubs and the University. Participants were included if they were male and had been actively participating in non-professional Australian football. Participants were asked to maintain a consistent diet in the 24 h leading into the testing days, including the discouragement of the use of caffeine. The participants were competing in various regional competitions of a similar standard (non-professional), completed at least 5 h of Australian football-specific activity per week (training and matches), and had a mean (±SD) stature of 180±5 cm, mass of 78.0±6.0 kg, and age of 20.7±1.6 years. Ethical approval was obtained from the University Human Research Ethics Committee (project approval number A11-002) and the study complied with the Declaration of Helsinki’s ethical guidelines. The risks were presented to each participant in writing and verbally prior to the study. Written informed consent was then collected from each participant. 2.2. Procedures The study had a correlational design, with subjects completing the protocols in a randomized, counter-balanced order. Each participant was required to complete the Yo-Yo IR2 test and metabolic capacity tests on separates days within 2 weeks of each other. The Yo-Yo IR2 test was completed on a stable indoor surface using procedures established previously [7]. Participants were familiarized with the testing protocols of Yo-Yo IR2 by participating in a sub-maximal Yo-Yo IR2 test up to 48 h prior to testing without exhaustion. These procedures have been found to be reliable and applicable to team sports such as soccer and Australian Rules football [5,7]. The metabolic tests were performed in an exercise physiology laboratory. The aerobic metabolic

testing without exhaustion. These procedures have been found to be reliable and applicable to team sports such as soccer and Australian Rules football [5,7]. The metabolic tests were performed in an exercise physiology laboratory. The aerobic metabolic

Sports2024,12, 236 3 of 8 performance capabilities assessed were peak oxygen uptake, maximal aerobic speed, and relative intensity (running economy). The anaerobic metabolic capacity was determined by the maximal accumulated oxygen method (MAOD) [16]. The MAOD protocols were familiarized to the participants with a non-exhaustive practice run. All participants indicated that they had used the laboratory equipment before for similar testing protocols. Participants were in pre-season and not competing at the time of testing; testing was performed on a day off from training for all participants at a time of their convenience. However, the Yo-Yo IR2 tests and metabolic tests were performed at a similar time of day for each individual. Participants were required to perform 2×20 m shuttles in time to an audio beep of progressively increasing intensity with 10 s of rest between shuttles, in which the participant jogged around a cone situated 5 m past the start/finish line. Each participant received a warning if they did not make the shuttle in time; upon missing it a second time, they were eliminated from the test. The total distance covered in meters during the test was deemed their Yo-Yo IR2 score in accordance with Krustrup et al. [7]. The MAOD protocol was conducted in a laboratory on a motorized treadmill ergome- ter at a 1% gradient (T200, Cosmed, Rome, Italy) to replicate the energy cost of over-ground running [17]. The participants ran at five progressively faster speeds for 5 min at each speed (8, 9.5, 11, 12.5, and 14 km·h −1 ) without a break between each speed for 25 min of accumulated running time. Before they ran, participants were fitted to a two-way breathing apparatus (Hans Rudolph, Shawnee, KS, USA) through which expired air was analysed by an online metabolic system (Moxus, AEI Technologies, Bastrop, TX, USA) previously calibrated with gases of known concentration (20.9% and 16% O2and 0.3% and 4% CO2) and volume (3 L syringe). The volume of oxygen consumption was recorded every 30 s and averaged for the final two min of each 5 min period. Continuing without rest from the final submaximal run, the

online metabolic system (Moxus, AEI Technologies, Bastrop, TX, USA) previously calibrated with gases of known concentration (20.9% and 16% O2and 0.3% and 4% CO2) and volume (3 L syringe). The volume of oxygen consumption was recorded every 30 s and averaged for the final two min of each 5 min period. Continuing without rest from the final submaximal run, the intensity was increased by 1 km·h −1 every minute until ventilatory exhaustion and VO2plateau to obtain VO 2peak. VO 2peakwas determined as the highest value recorded in 30 s intervals and reflects the highest aerobic metabolic rate of the individ- ual. Submaximal relative intensity was calculated to be the average VO2(mL·kg −1 · min −1 ) during the final two min of the 12.5 km·h −1 stage, presented as a percentage of VO 2peak. Running economy was determined as the actual steady state VO2(mL·kg −1 · min −1 ) during the 12.5 km·h −1 stage. The mean VO2values for the final two minutes of each of the five submaximal run speeds were submitted to a linear regression model to determine the supra-maximal treadmill speed required at 115% of VO 2peak. The coefficient was very high, ranging from r 2 = 0.93 to 0.99, similar to prior work (16). The VO 2peakof each participant was substituted into the regression equation to determine their predicted maximal aerobic speed (MAS), defined as the speed corresponding to VO 2peak.Participants were given 35 min of passive rest before completing the supra-maximal run to exhaustion; they were encouraged to drink fluids ad libitum and restricted to ingesting less than 20 g of carbohydrate to avoid possible gastrointestinal distress. Participants ran at a speed calculated to reflect 115% of VO 2peak on a 1% gradient until exhaustion. Participants waited until the treadmill reached the prescribed speed, then lowered themselves onto the treadmill belt. Data collection began when the participant removed their hands from the rails of the treadmill. Collection ceased once the participant indicated they could no longer maintain the speed either by pressing a stop button on the side of the treadmill or by a non-verbal

Participants waited until the treadmill reached the prescribed speed, then lowered themselves onto the treadmill belt. Data collection began when the participant removed their hands from the rails of the treadmill. Collection ceased once the participant indicated they could no longer maintain the speed either by pressing a stop button on the side of the treadmill or by a non-verbal signal to the test administrators. The participants were fitted with a two-way breathing apparatus, with expired air be- ing collected directly by Douglas bags and analysed using first principles [16]. The expired air was pumped into a metabolic system to determine the O2and CO2concentrations at a rate of 3 L per min; the extraction was timed, and the residual volume was included in the calculations. Expired air was then extracted manually by a 2 L syringe until the bags were empty to determine the total volume of air. The volume of air was then standardized for pressure, water content, temperature, and N2content. VO2was then calculated as below (Equation (1)). VO2I was calculated using the Haldane transformation, whilst VO2E was calculated from the percentage of O2content multiplied by the standardized volume.

Sports2024,12, 236 4 of 8 VO2= VO2I−VO2E (1) •VO2= Oxygen uptake. •VO2I = Volume of oxygen inspired. •VO2E = Volume of oxygen expired. MAOD was then calculated as the predicted VO2subtracted from the actual VO2, as previously described [16,18–21]. The duration of the supra-maximal run was also recorded. Each participant was scheduled for an hour in the laboratory to complete testing. 2.3. Statistical Analyses Prior to the study, a power calculation was performed to determine the sample size required to detect an r of 0.6 with a power of 0.8 and a statistical significance of 0.05 in a two-tailed test. Nineteen participants were identified and recruited. Following the testing procedures, all data underwent statistical analyses using SPSS (version 26; IBM, Armonk, NY, USA). Normality was assessed using the Shapiro–Wilk test and via the visual inspection of histograms and Q-Q plots. Descriptive statistics (mean and standard deviation) were calculated for each variable. The data were separated into high (n= 9) and low (n= 10) Yo-Yo IR2 scores using the median split technique. All variables were then analysed for differences between high and low Yo-Yo IR2 groups using a one-way ANOVA. Partial correlations were also used to isolate the contribution of variables independent of other associated variables (found in the correlation matrix) [22]. The significance level for all statistical tests was set atp< 0.05. 3. Results The Yo-Yo IR2 test revealed the participants’ mean (±SD) distance to be689±155 m, ranging between 440 m and 920 m. Furthermore the treadmill protocol revealed that the participants had a mean VO 2peakof 56.5±5.9 mL·kg −1 · min −1 , a relative intensity of 84.1±7.9%, a VO 2peakof 12.5 km·h −1 , a running economy of 12.5 km·h −1 47.3±3.7 mL· kg −1 · min −1 , a predicted MAS of 15.0±1.4 km·h −1 , an MAOD of 13.7±6.0 mL O2 eq·kg −1 · min −1 , and time to exhaustion at 115% MAS of181.5±53.1 s . A correlation matrix between the measured variables is also presented in Table. These results show significant relationships between Yo-Yo IR2 performance and VO 2peak(r = 0.62,p= 0.005), relative intensity

, a predicted MAS of 15.0±1.4 km·h −1 , an MAOD of 13.7±6.0 mL O2 eq·kg −1 · min −1 , and time to exhaustion at 115% MAS of181.5±53.1 s . A correlation matrix between the measured variables is also presented in Table. These results show significant relationships between Yo-Yo IR2 performance and VO 2peak(r = 0.62,p= 0.005), relative intensity (% VO 2peakat 12.5 km·h −1 ) (r =−0.72,p= 0.001), and predicted MAS (r = 0.70,p= 0.001). However, no significant relationship was revealed between running economy and Yo-Yo IR2 performance (r = 0.05,p= 0.86). When separated into high and low Yo-Yo IR2 groups, the mean Yo-Yo IR2 performance was 738±102 m (ranging 640–920 m) for the high group and 492±62 m (ranging 440–600 m) for the low group. There was a significant difference in VO 2peak, relative intensity, and predicted MAS between the high and low Yo-Yo IR2 groups (Table). Table 1.Correlation matrix between measured variables. MAOD Relative Intensity Running Economy Predicted MAS Yo-Yo IR2 (m) VO 2peak (mL·kg −1 ·min −1 ) r 0.70 −0.70 0.48 0.54 0.62 p0.001 0.001 0.04 0.02 0.005 MAOD (mL O 2eq·kg −1 ·min −1 ) r −0.22 0.66 0.04 0.16 p 0.36 0.002 0.89 0.50 Relative Intensity (%VO 2at 12.5 km·h −1 ) r 0.29 −0.91 −0.72 p 0.22 0.00 0.001 Running Economy (VO 2mL·kg·min −1 at 12.5 km·h −1 ) r −0.40 −0.05 p 0.09 0.86 Predicted MAS (km·h −1 ) r 0.70 p 0.001 r = Pearson’s correlation coefficients. MAOD = maximal accumulated oxygen deficit. MAS = maximal aerobic speed.

Sports2024,12, 236 5 of 8 Table 2.The difference in variables between high and low Yo-Yo IR2 groups (mean±SD, 95% confidence limits in parentheses). High Yo-Yo IR2 Low Yo-Yo IR2 Difference in Mean pValue VO 2peak (mL·kg −1 ·min −1 ) 60.13±4.3 53.93 ±5.66 6.2 (1.1–11.3) 0.02 MAOD (mL O 2eq·kg −1 ·min −1 ) 15.55±6.71 12.43 ±5.26 3.1 ( −2.7–8.9) 0.27 Relative Intensity (%VO 2at 12.5 km·h −1 ) 78.93±4.57 87.92 ±7.83 −9.0 (−15.5–−2.4) 0.01 Running Economy (VO 2mL·kg −1 ·min −1 at 12.5 km·h −1 ) 47.39±3.43 47.16 ±4.1 0.22 ( −3.5–4.0) 0.9 Predicted MAS (km·h −1 ) 15.84±1.23 14.38 ±1.24 1.5 (0.2–2.7) 0.02 High (n= 8) and low (n= 11) Yo-Yo IR2 scores. MAOD = maximal accumulated oxygen deficit. MAS = maximal aerobic speed. Figure VO 2peakand relative intensity, predicted MAS is not significantly related to Yo-Yo IR2 performance (r = 0.28,p= 0.28). However, when predicted MAS was controlled, VO 2peak and Yo-Yo IR2 performance showed a trend towards a relationship (r = 0.46,p= 0.095).Sports 2024, 12, x FOR PEER REVIEW 6 of 9 Figure 1. Partial correlation matrix showing the interaction between variables when controlling for other related variables. The solid lines represent the interaction whilst the dotted arrows represent the variable controlling for the interaction. Pearson’s correlation coefficients are represented in this figure with significance at the 0.05 level shown by asterisks. 4. Discussion This study aimed to identify the aerobic and anaerobic metabolic and performance capacities contributing to Yo-Yo Intermittent Recovery level 2 (Yo-Yo IR2) test perfor- mance. The results revealed that predicted maximal aerobic speed (MAS) and relative in- tensity had the strongest associations with Yo-Yo IR2 performance. Additionally, when participants were grouped based on their Yo-Yo IR2 scores, significant differences in MAS, relative intensity, and VO 2peak were observed. Further analyses uncovered a series of direct and indirect interactions between all measured variables, which can be used to identify areas of weakness in an athlete’s physical performance in sports where the Yo-Yo IR2 test is a valid measure. Overall, the findings highlight the intricate interplay between endur- ance qualities and Yo-Yo IR2 performance

intensity, and VO 2peak were observed. Further analyses uncovered a series of direct and indirect interactions between all measured variables, which can be used to identify areas of weakness in an athlete’s physical performance in sports where the Yo-Yo IR2 test is a valid measure. Overall, the findings highlight the intricate interplay between endur- ance qualities and Yo-Yo IR2 performance and suggest that a comprehensive approach is required to optimize athletic performance in such sports. Additionally, there was a signif- icant correlation between Yo-Yo IR2 performance and VO 2peak. Improvements in VO 2peak have been shown to be coupled with improvements in Yo- Yo IR1 test performance and other repeated-sprint ability tests in previous training studies [23–26]. However, it is not clear whether the differences in this study were due to VO 2peak alone or due to the accumulation of effects of MAS and running economy. The results showed that running economy and maximal accumulated oxygen de ficit (MAOD) showed indirect associations with Yo-Yo IR2 by correlating with VO 2peak and relative in- tensity. Figure 1.Partial correlation matrix showing the interaction between variables when controlling for other related variables. The solid lines represent the interaction whilst the dotted arrows represent the variable controlling for the interaction. Pearson’s correlation coefficients are represented in this figure with significance at the 0.05 level shown by asterisks.

The study investigates metabolic capacities affecting Yo-Yo IR2 test performance in Australian footballers.