The Positive Effects of Priming Exercise on Oxygen Uptake Kinetics and High-Intensity Exercise Performance Are Not Magnified by a Fast-Start Pacing Strategy in Trained Cyclists

The purpose of this study was to determine both the independent and additive effects of prior heavy-intensity exercise and pacing strategies on the VO2 kinetics and performance during high-intensity exercise. Fourteen endurance cyclists (VO2max  = 62.8±8.5 mL.kg−1.min−1) volunteered to participate in the present study with the following protocols: 1) incremental test to determine lactate threshold and VO2max; 2) four maximal constant-load tests to estimate critical power; 3) six bouts of exercise, using a fast-start (FS), even-start (ES) or slow-start (SS) pacing strategy, with and without a preceding heavy-intensity exercise session (i.e., 90% critical power). In all conditions, the subjects completed an all-out sprint during the final 60 s of the test as a measure of the performance. For the control condition, the mean response time was significantly shorter (p<0.001) for FS (27±4 s) than for ES (32±5 s) and SS (32±6 s). After the prior exercise, the mean response time was not significantly different among the paced conditions (FS = 24±5 s; ES = 25±5 s; SS = 26±5 s). The end-sprint performance (i.e., mean power output) was only improved (∼3.2%, p<0.01) by prior exercise. Thus, in trained endurance cyclists, an FS pacing strategy does not magnify the positive effects of priming exercise on the overall VO2 kinetics and short-term high-intensity performance.


Introduction
Exercise intensity domains (i.e., moderate, heavy and severe) are defined according to the blood lactate and oxygen uptake (VO 2 ) responses obtained during constant-work-rate exercise [1]. Critical power (CP -the asymptote of the power-time relationship) is considered the lower boundary of the severe-intensity domain [2]. Indeed, during constant-work-rate exercise performed within the severe domain, the VO 2 rises inexorably (as the slow component of the VO 2 kinetics increases) to the maximal oxygen uptake (VO 2 max). Exercise tolerance within the severe domain can be predicted and is defined by the curvature constant of the powertime relationship (W9) [3]. Several lines of evidence indicate that the interaction between VO 2 kinetics, W' and the attainment of VO 2 max can contribute to exercise intolerance during exercise performed in the severe-intensity domain [4]. Some interventions (e.g., pacing, priming exercise and nitrate supplementation) that are used to improve VO 2 kinetics (i.e., t -the time taken to reach 63% of the increase in VO 2 above baseline and/or the slow component of VO 2 kinetics) can reduce the W' utilization during the initial phase of exercise, improving performance [5] and exercise tolerance [6] during severe-intensity exercise.
Pacing strategy (i.e., the pattern of the rate of energy expenditure) has important effects on exercise tolerance [7] and performance [5]. The self-selected pacing strategy adopted during a time trial is controlled by a complex regulatory system, in which integrated neural control regulates exercise intensity to prevent homeostatic disturbances that might cause injury [8]. Factors such as exercise modality, event duration and performance level can influence the self-selected pacing strategy [9,10]. Some studies have demonstrated that a fast-start pacing strategy has a positive effect on performance during sports events of up to approximately 2-3 min in duration [11,12]; in these events, energy is provided by both aerobic and anaerobic pathways [13]. In these conditions, the VO 2 kinetics is significantly faster, sparing W' utilization during the initial phase of exercise [5,7]. Interestingly, Jones et al. [7] found that the percentage reduction in the mean response time of VO 2 was significantly correlated (r = 0.85, p,0.05) with the percentage improvement in exercise tolerance when a fast start was compared with an even-paced exercise.
Warm-up exercise has been extensively performed by athletes before their participation in subsequent vigorous exercise. Indeed, priming exercise performed at heavy or severe intensities domain can improve exercise tolerance during severe-intensity exercise (submaximal and perimaximal) [6,14]. These positive alterations have been attributed, at least in part, to enhancement of the overall VO 2 kinetics [6,15]. Gerbino et al. [16] and MacDonald et al. [17] demonstrated that prior heavy exercise accelerated the monoexponential kinetics (i.e., mean response time) during a second bout of heavy exercise performed 6 min after the first bout. Later, studies using a more comprehensive model (two or three components) to analyze VO 2 kinetics [6,18] demonstrated that this overall acceleration could be attributed to the increased amplitude of the primary component and the reduced amplitude of the slow component, with the time constant of the primary component (i.e., t) remaining unaffected. A similar response (i.e., increased amplitude of the primary component and unchanged t) was found for exercise that was performed at perimaximal intensities (100%, 110% and 120% of VO 2 max) after prior heavy exercise [14].
Thus, the interventions discussed above (i.e., priming exercise and pacing) seem to have different effects on the VO 2 kinetics during severe-intensity exercise. The mechanism that underpins these effects is unclear. However, it is possible that different factors contribute to the VO 2 response profile under these conditions (pacing vs. priming exercise). Priming exercise seems to increase blood flow, oxygenation, oxidative enzyme activity and electromyographic activity, thus accelerating the overall VO 2 response to severe exercise [15]. A positive pacing strategy, which causes a higher initial rate of muscle ATP hydrolysis, can magnify the VO 2 ''error signal'', i.e., the difference between the instantaneous supply and the required rates of oxidative phosphorylation [5]. The absolute rate at which VO 2 increases after the onset of exercise is a positive function of the ''error signal'' [19]; therefore, a fast-start pacing strategy results in faster VO 2 kinetics [5,7]. Given this scenario, it is possible that priming exercise can amplify the positive effect of a fast-start pacing strategy on VO 2 kinetics and exercise tolerance/performance during high-intensity exercise. However, the possible additive effects of priming exercise and pacing strategy on these variables are unknown.
The purpose of this study was to determine the independent and additive effects of prior heavy-intensity exercise and pacing strategies on VO 2 kinetics and performance during high-intensity exercise. The following hypotheses were proposed: 1) A fast-start pacing strategy would shorten the mean response time, and increase peak power output and mean power output during shortterm high-intensity exercise; and 2) Priming exercise would shorten the mean response time, and increase peak power output and mean power output during short-term high-intensity exercise irrespectively of the utilized pacing strategy.

Ethics statement
The present study was approved by the Ethics Committee of the Biosciences Institute -Rio Claro of São Paulo State University, and all subjects provided written informed consent prior to participation. The study was performed in accordance with the declaration of Helsinki.

Subjects
Fourteen endurance cyclists (2665 years; 7169 kg; 17568 cm) with at least 5 years of experience in the modality volunteered to participate in the present study; these athletes were competing in regional-to national-level meets. The subjects were familiar with the laboratory testing procedures, as they were previously involved in similar evaluations. The subjects were instructed to be fully rested and hydrated at least 3 h postprandially when reporting to the laboratory and to refrain from using caffeine-containing food or beverages, drugs, alcohol, cigarette, or any form of nicotine 24 h before testing. Each subject was tested in a climate-controlled (21-22uC) laboratory at the same time of day (62 h) to minimize the effects of diurnal biological variation.

Experimental design
The subjects were required to visit the laboratory on 11 different occasions, separated by at least 24 h, within a period of three weeks. The first visit to the laboratory was to undergo an incremental test to determine the lactate threshold, VO 2 max and the power output at VO 2 max (PVO 2 max). On the following four visits, the subjects underwent four constant-load tests (75%, 80%, 85% and 100% of PVO 2 max) to exhaustion, in random order, to determine the parameters of the power-duration relationship (i.e., CP and W9). The CP model was used to estimate the workload that would be expected to lead to exhaustion in 3 min (P3-min). From the 6 th to the 11 th visit, the subjects performed three different pacing strategies (fast start, even start, and slow start) with and without a preceding heavy-intensity exercise session.

Incremental protocol
The incremental protocol was performed on a cycle ergometer (Lode Excalibur Sport, Lode BC, Groningen, Netherlands) with the subjects pedaling at a constant self-selected pedal rate (between 70 and 90 rpm). The chosen pedal rate along with saddle and handle bar height and configuration was recorded and reproduced in subsequent tests. The initial power output was 120 W for 3 min and was then increased by 20 W every 3 min. Capillary blood samples were collected within the final 20 s of each stage for the determination of the blood lactate concentration ([La]). The [La] were determined (YSI 2300, Yellow Springs, Ohio, USA) immediately and the test was stopped when the [La] rose above 4 mM. Plots of [La] against the power output were provided by two independent reviewers, who determined the lactate threshold as the first sudden and sustained increase in blood lactate above resting concentrations [20]. After a rest period of 30 min, the participants performed a fast ramp test. The test began with an initial 5 min of cycling at 25 W below their previously determined lactate threshold, and the power was subsequently increased by 5 W every 12 s until voluntary exhaustion. The protocol was terminated when a drop of more than 5 rpm of their self-selected cadence occurred for more than 5 seconds despite strong verbal encouragement. VO 2 max was defined as the highest average 15-s VO 2 value recorded during the incremental test. Pulmonary gas exchange was measured continuously using a breath-by-breath analyzer (Cosmed Quark PFTergo, Rome, Italy). Before each test, the O 2 and CO 2 analysis systems were calibrated using ambient air and a gas of known O 2 and CO 2 concentration according to the manufacturer's instructions, while the gas analyzer turbine flowmeter was calibrated using a 3-L syringe. The heart rate was also monitored throughout the tests (Polar, Kempele, Finland). The PVO 2 max was defined as the power output at which VO 2 max occurred. The work rate that would require 50%D (work rate at the lactate threshold plus 50% of the difference between the work rate at the lactate threshold and VO 2 max) was subsequently calculated.

Determination of the power-duration relationship
The exercise protocol began with a 10 min warm-up at lactate threshold, followed by 5 min of rest prior to the commencement of the exhaustive trial [21]. Thereafter, the subjects exercised for 3 min at 20 W followed by a constant-workload test (75%, 80%, 85% and 100% of PVO 2 max) to voluntary exhaustion or until the subject could not maintain the required cadence (i.e., a cadence ,5 rpm of the preferred cadence) despite verbal encouragement [21]. These tests were conducted at the same cadence as the incremental test. During these testing sessions, the participants were not informed of the imposed work rate, their performance times or their heart rate. The exercise tolerance (tlim) was measured to the nearest second. The three equivalents of the 2parameter model [P = (W9/tlim)+CP; tlim = W9/(P-CP); W = CP?tlim+W9] were used to fit the data and estimate CP and W9 [22] using an iterative nonlinear regression procedure (Microcal Origin 7.5; Northampton, MA, USA) for each subject. The CP and W9 estimates from the 3 equations were compared to select the best fit using the model associated with the lowest standard error for CP (SEE) [23,24].

Experimental sessions
The exercise protocol began with a 5 min warm-up at lactate threshold, followed by 7 min of rest. Thereafter, the subjects performed 3 min at 20 W before the experimental conditions. In the even-start (ES) condition, the athletes performed 2 min of constant-load exercise at P3-min, followed by a 1-min all-out exercise period. In the fast-start (FS) condition, the first 90 s of exercise was performed as the work rate was reduced linearly from 110% to 90% of P3-min, followed by a 1-min all-out exercise period. In the slow-start (SS) condition, the first 90 s of exercise was performed as the work rate was increased linearly from 90% to 110% of P3-min, followed by a 1-min all-out exercise period. The last 30 s of the FS and SS conditions was performed at P3min [5]. During the first 2 min of exercise, a hyperbolic mode was used (fixed power), which was immediately changed to a linear mode (power dependent on the cadence) during the all-out exercise. These experimental conditions were performed in random order with and without previous exercise ( Figure 1).

1-min all-out exercise
Following the 2-min pacing exercises, the athletes performed 1 min of all-out exercise. They were required to reach the peak power as quickly as possible and to exert maximal effort during the whole test. Throughout the 1-min test, the athletes were given verbal encouragement but were not informed of time elapsed. The VO 2 was measured breath-by-breath during the exercise, and the data were reduced to 15-s stationary averages. The resistance to pedaling was calculated using the preferred cadence obtained during the incremental test and the workload corresponding to 50%D: The following performance data were obtained from the all-out test: peak power output, time to peak power output and mean power output.

Prior exercise
The prior exercise conditions involved participants performing 3 min of baseline cycling at 20 W, followed by a square-wave transition to a work rate requiring 90% CP (i.e., heavy-intensity exercise). At 6 min, the subjects were allowed to ''spin down'' against zero resistance for 1 min and then rested passively for 6 min before remounting the ergometer and pedaling for 3 min at 20 W. After this 3-min period, one of the three pacing conditions was immediately imposed as described above. One minute before and immediately after these exercise bouts, a fingertip capillary blood sample was taken to determine the blood [La]. The subjects repeated this process on separate days and in a randomized order until all experimental trials were completed.

VO 2 kinetics
The breath-by-breath data from each exercise test were filtered manually to remove outlying breaths, which were defined as breaths deviating by more than four standard deviations from the preceding five breaths. The breath-by-breath data were subsequently linearly interpolated to provide second-by-second values and aligned by time to the start of the exercise, and a nonlinear least squares algorithm was used to fit the data thereafter. A singleexponential model without a time delay and with a fitting window commencing at t = 0 s (equivalent to the mean response time) was used to characterize the kinetics of the overall VO 2 response during initial phase (i.e., 90 s) of the different pacing strategies for all subjects. The following equation describes this model: where VO 2 (t) represents the absolute VO 2 at a given time t, VO 2 baseline represents the mean VO 2 measured over the final 60 s of baseline pedaling, and A and t represent the amplitude and time constant, respectively, which describe the overall increase in VO 2 above the baseline. The oxygen deficit was also calculated for the same time period (i.e., 90 s) by multiplying the mean response time and the DVO 2 .

Statistical analysis
The data are reported as the means 6SD. The normality of data was checked by the Shapiro-Wilk test. The data were analyzed using two-way ANOVA (prior exercise vs. pacing strategy), with Fisher's LSD test where appropriate. For all statistics, the significance level was set at p#0.05.

Results
During the ramped incremental test, the subjects attained a peak work rate (i.e., PVO 2 max) of 411645 W, a VO 2 max of 4.4360.47 L.min 21

Discussion
The purpose of this study was to determine the independent and additive effects of prior heavy-intensity exercise and pacing strategies on VO 2 kinetics and performance during high-intensity exercise. Similar to previous studies, we have demonstrated that both priming exercise [6] and pacing strategies (i.e., FS) [5,7] accelerated the overall VO 2 kinetics (i.e., mean response time). However, our study reveals, for the first time, that an FS pacing strategy does not magnify the positive effects of prior heavyintensity exercise on the overall VO 2 kinetics. Moreover, the performance during high-intensity exercise (i.e., peak power output and mean power output) was enhanced only by prior heavy-intensity exercise. These data confirm and extend the proposal that the changes (i.e., speeding/slowing) in the VO 2 kinetics during the initial phase of different pacing strategies (FS, ES and SS) are not necessarily associated with the changes in performance during short-term high-intensity exercise [5].
Some studies have found that the overall VO 2 kinetics is accelerated by an FS pacing strategy when compared with ES and SS strategies [5,7]. Factors such as exercise modality [5,11] and aerobic performance level [6] do not appear to influence the effects of the FS pacing strategy on the overall VO 2 kinetics. Thus, our data confirm that an FS pacing strategy can improve the overall VO 2 kinetics during high-intensity exercise in trained endurance cyclists. Studies have shown a direct proportionality between the products of PCr splitting and muscle or pulmonary VO 2 [19]. An FS pacing strategy requires a greater initial rate of muscle ATP hydrolysis, resulting in a greater initial D [PCr]/D time. Thus, a more rapid accumulation of the metabolites (ADP, Pi and Ca 2+ ) that stimulate oxidative phosphorylation would be subject's VO 2 max. Panels A, B and C -slow start, even start and fast start pacing conditions, respectively. Grey circles and black circles -paced exercise trials, with and without prior exercise, respectively. Notice that the VO 2 response is speeded using the fast start pacing strategy only in the control condition. Thus, the fast start pacing strategy does not magnify the positive effects of prior heavy-intensity exercise on overall VO 2 response. doi:10.1371/journal.pone.0095202.g002 observed during an FS pacing strategy. Accordingly, Bailey et al. [5] used near-infrared spectroscopy (NIRS) to verify that FS strategies might be linked to increased muscle O 2 extraction.
The classic experiments of Gerbino et al. [16] demonstrated that prior heavy exercise accelerated the monoexponential kinetics (i.e., mean response time) during a second bout of heavy exercise performed 6 min after the first. Later, studies using different experimental designs (e.g., intensities, durations of recovery time and age group) [6,25] confirmed the seminal results obtained by Gerbino et al. [16]. Our experimental results revealed that both the mean response time and VO 2 amplitude (the overall increase in VO 2 above the baseline) were modified by previous heavy exercise. The increased VO 2 amplitude during a second bout of severe exercise has been considered important for exercise tolerance/performance, because the slow component of the VO 2 kinetics, the change in blood lactate concentration and the aerobic contribution are positively modified during the second bout of exercise. Central (increases in bulk O 2 delivery) and peripheral (convective O 2 delivery and increased activity of mitochondrial enzymes) factors are possible explanations for this altered VO 2 response profile during the second bout of exercise [15].
To the best of our knowledge, this study is the first to determine the possible additive effects of priming exercise and pacing strategy on VO 2 kinetics during severe-intensity exercise. We have demonstrated that previous heavy-intensity exercise accelerated the overall VO 2 kinetics only during SS and ES pacing strategies. Moreover, there was no significant difference among the FS, ES and SS preceded by previous heavy-intensity exercise. Thus, the effects of priming exercise on VO 2 kinetics during severe-intensity exercise are dependent on pacing strategy. Moreover, these effects do not appear to be magnified by an FS pacing strategy. Together, these results suggest that previous exercise has great potential to enhance the overall VO 2 kinetics and that an FS pacing strategy does not amplify its effects.
In the present study, we did not use a biexponential model to characterize the VO 2 kinetics because we were unable to repeat each trial to enhance the signal-to-noise ratio of the VO 2 responses (see below). Thus, the parameters of the VO 2 kinetics were not characterized. However, the exercise intensity used during the different pacing strategies was similar to PVO 2 max; therefore, the VO 2 slow component, that elevates the VO 2 above the steadystate value predicted from the sub-lactate threshold VO 2 -work rate relationship [26], cannot be detected under these circumstances. Thus, in line with other studies, the previous exercise may have only enhanced the VO 2 amplitude [15], while the pacing strategy enhanced the time constant of the primary component of the VO 2 response [5,7]. Nevertheless, previous heavy-intensity exercise blunted the effects of the FS pacing strategy on the overall VO 2 kinetics. Therefore, the alterations caused by previous exercise (available O 2 , convective O 2 delivery, activity of mitochondrial enzymes and motor unit recruitment) appear to prevent the effects of an FS pacing strategy on the VO 2 response. In line with this statement, Rossiter et al. [27] have found that prior high-intensity exercise reduced the amplitude of the [PCr] response, with the initial rate of [PCr] change (d[PCr]/dt) remaining unaffected during a second bout of heavy exercise. These alterations are suggestive of a reduced t[PCr] during primed exercise [27], although the difference between conditions (i.e., control, 34 s vs. primed exercise, 32 s) did not reach statistical significance. Moreover, it has been demonstrated that the intramuscular enzyme activity status (i.e., pyruvate dehydrogenase complex -PDC), can allow a greater flux of acetyl groups into the mitochondria for oxidation [28]. An increased activation of pyruvate dehydrogenase complex might reduce both substratelevel phosphorylation (i.e., glycolysis and the creatine kinase and adenylate kinase reactions) [28] and the primary component time  constant [29]. Thus, these mechanisms (altered [PCr] kinetics and/or increased PDC activity) might have blunted the effects of an FS pacing strategy on the VO 2 response (i.e., VO 2 ''error signal''). However, future studies, with appropriated experimental design, should be conducted to confirm (or not) these hypotheses. Previous heavy-or severe-intensity exercises have been shown to improve exercise tolerance during both submaximal [6] and perimaximal exercise [14]. This positive effect seems to be influenced by an optimal interaction between prior exercise intensity and recovery duration [6]. We have provided the first demonstration that prior heavy-intensity exercise enhances performance (peak power output and mean power output), and this increase occurs independently of the chosen pacing strategy. Improved exercise tolerance during submaximal intensity has been observed when the amplitude of the slow component of the VO 2 kinetics decreased and the overall VO 2 kinetics were faster [6]. Indeed, we have observed that the overall VO 2 kinetics was faster and the VO 2 amplitude increased (and thus the magnitude of the O 2 deficit was reduced) after heavy-intensity exercise. Thus, previous heavy-intensity exercise can reduce the W9 utilization during the initial phase of exercise, improving performance during short-term high-intensity exercise.
It has been proposed that the mild lactic acidosis caused by prior heavy exercise might increase oxygen delivery by stimulating vasodilatation and a rightward shift in the oxyhaemoglobin dissociation curve (i.e. the Bohr effect) [16]. Based on data obtained in active subjects (VO 2 max,50 mL.kg 21 .min 21 ), some studies have suggested that a baseline blood lactate concentration of ,3 mM results in an increased time to exhaustion during subsequent high-intensity exercise [6,14]. Moderate-intensity prior exercise, which did not alter the baseline blood lactate concentration, does not enhance VO 2 kinetics or exercise tolerance during subsequent high-intensity exercise performed by active subjects [16]. Similarly, Bailey et al. [6] have shown that the effect of prior heavy exercise on VO 2 kinetics is prevented when baseline blood [lactate] recovers to ,2 mM. However, we have verified that a baseline blood lactate concentration of ,1.8 mM has enhanced both overall VO 2 kinetics and short-term high-intensity performance in trained endurance cyclists. The low blood lactate concentration found 9 min after heavy intensity exercise can be explained, at least in part, by increased rate of blood lactate removal found in aerobic trained athletes [30]. Interestingly, Burnley et al. [31] have found that moderate-intensity prior exercise enhanced both primary VO 2 amplitude and exercise performance in well-trained cyclists (VO 2 max ,58 mL.kg 21 .min 21 ). Thus, in aerobic trained cyclists, it seems that the presence of an elevated blood lactate concentration is not a sine qua non condition for improving VO 2 kinetics and short-term high-intensity performance after prior exercise.
Some studies found that an FS pacing strategy can improve exercise tolerance [7] and performance [5] during short-term high-intensity exercise. In the present study, the pacing strategy did not significantly influence the exercise performance, although the overall VO 2 kinetics was improved by the FS pacing strategy. Some interventions (priming exercise and pacing) have shown similar results [5,6], indicating that changes in the overall VO 2 kinetics will not necessarily enhance exercise tolerance/perfor-mance during subsequent high-intensity exercise. Interestingly, Bailey et al. [5] reported that utilizing an FS pacing strategy with active individuals (VO 2 max ,52 mL.kg 21 .min 21 ) improved both the overall VO 2 kinetics and exercise performance during subsequent high-intensity exercise. In the present study, we analyzed trained endurance cyclists (VO 2 max = 62 mL.kg 21 .min 21 ). Thus, differences in aerobic fitness might explain, at least in part, these different results. Bailey et al. [5] proposed that the attainment of VO 2 max during high-intensity exercise bouts, when this is ordinarily not possible, is essential for improving exercise performance. Given the finite speed of the VO 2 response, the exercise durations at the extreme domain [32] would be too short to permit attainment of VO 2 max [21]. Thus, the attainment of VO 2 max would allow a more complete depletion of W9 and consequently allow better exercise performance [5]. Indeed, we have verified that VO 2 max was not attained during the FS pacing strategy. However, future studies using different experimental designs should be conducted to test this relationship.
Because of the nature of the present experiments, certain limitations of the study should be considered when interpreting its findings. The determination of the VO 2 response parameters in the heavy-and severe-intensity domain using only one transition can have potential limitations (i.e., low confidence in the response parameters). Repeated bouts have traditionally been averaged to improve the signal-to-noise ratio of data [33]. However, due to the extremely demanding nature of the exercise testing and the frequent laboratory visits (11), only one trial was conducted for each experimental condition. Although we only measured one transition, the signal-to-noise ratio of the data can be improved by using higher VO 2 amplitudes [33]. Therefore, higher VO 2 amplitudes, as utilized in the present study, correspond to smaller confidence intervals. Indeed, the 95% confidence interval for the estimation of mean response time was ,3 s for all conditions (Table 1).
In summary, we have demonstrated in trained endurance cyclists that priming heavy-intensity exercise has a positive effect on both overall VO 2 kinetics and short-term high-intensity performance. However, the FS pacing strategy only modified the overall VO 2 kinetics. This finding suggests that faster overall VO 2 kinetics does not, per se, determine the performance (i.e., peak power output and mean power output) during high-intensity exercise. The FS pacing strategy does not magnify the positive effects of prior heavy-intensity exercise on the overall VO 2 kinetics. Thus, the modifications caused by priming exercise preclude the effects of the FS pacing strategy on the overall VO 2 kinetics. Finally, priming exercise seems to have greater potential than FS pacing strategies to enhance both overall VO 2 kinetics and short-term high-intensity performance in trained endurance cyclists.