Vol.:(0123456789)
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European Journal of Applied Physiology (2021) 121:3083–3093 
https://doi.org/10.1007/s00421-021-04763-9
ORIGINAL ARTICLE
Training status affects between‑protocols differences 
in the assessment of maximal aerobic velocity
Andrea Riboli1  · Susanna Rampichini1  · Emiliano Cè1  · Eloisa Limonta1  · Marta Borrelli1  · 
Giuseppe Coratella1  · Fabio Esposito1,2 
Received: 15 February 2021 / Accepted: 2 July 2021 / Published online: 28 July 2021 
© The Author(s) 2021
Abstract
Purpose Continuous incremental protocols (CP) may misestimate the maximum aerobic velocity  (Vmax) due to increases in 
running speed faster than cardiorespiratory/metabolic adjustments. A higher aerobic capacity may mitigate this issue due to 
faster pulmonary oxygen uptake ( ̇VO2) kinetics. Therefore, this study aimed to compare three different protocols to assess 
 Vmax in athletes with higher or lower training status.
Methods Sixteen well-trained runners were classified according to higher (HI) or lower (LO) ̇VO2max ̇VO2-kinetics was 
calculated across four 5-min running bouts at 10 km·h−1. Two CPs [1 km·h−1 per min (CP1) and 1 km·h−1 every 2-min 
(CP2)] were performed to determine  Vmax ̇VO2max, lactate-threshold and submaximal ̇VO2/velocity relationship. Results were 
compared to the discontinuous incremental protocol (DP).
Results Vmax, ̇VO2max, ̇VCO2 and VE were higher [(P < 0.05,(ES:0.22/2.59)] in HI than in LO. ̇VO2-kinetics was faster 
[P < 0.05,(ES:-2.74/ − 1.76)] in HI than in LO. ̇VO2/velocity slope was lower in HI than in LO [(P < 0.05,(ES:-1.63/ − 0.18)]. 
 Vmax and ̇VO2/velocity slope were CP1 > CP2 = DP for HI and CP1 > CP2 > DP for LO. A lower [P < 0.05,(ES:0.53/0.75)] 
 Vmax-difference for both CP1 and CP2 vs DP was found in HI than in LO.  Vmax-differences in CP1 vs DP showed a large 
inverse correlation with  Vmax, ̇VO2max and lactate-threshold and a very large correlation with ̇VO2-kinetics.
Conclusions Higher aerobic training status witnessed by faster ̇VO2 kinetics led to lower between-protocol  Vmax differences, 
particularly between CP2 vs DP. Faster kinetics may minimize the mismatch issues between metabolic and mechanical power 
that may occur in CP. This should be considered for exercise prescription at different percentages of  Vmax.
Keywords ̇VO2 kinetics · Maximal aerobic power · Maximum oxygen uptake · Incremental test · Running velocity · 
Aerobic capacity
Abbreviations
HI 
 Group with high ̇VO2max
LO 
 Group with low ̇VO2max
CP1 
 Continuous incremental protocol 
[1 km·h-1 per min]
CP2 
 Continuous incremental protocol 
[1 km·h-1 every 2 min]
DP 
 Discontinuous incremental protocol
̇VO2max 
 Maximum oxygen uptake
̇VO2/Velocity slope  Regression analysis of the ̇VO2 vs 
velocity relationship at submaximal 
workloads
̇VO2 kinetics 
 ̇VO2-transition from rest to 
steady-condition
Vmax 
 The velocity associated with maxi-
mum oxygen uptake
̇VCO2 
 Carbon dioxide production
RER 
 Respiratory exchange ratio
SaO2 
 Arterial  O2 saturation
̇VE 
 Expiratory ventilation
BLa- 
 Blood lactate concentration
RPE 
 Rate of perceived exertion
ANOVA 
 Analysis of variance
ES 
 Effect size
95% CI 
 95% Confidence intervals
Communicated by Guido ferrati.
 * Andrea Riboli 
 
riboliandrea@outlook.com
1 
Department of Biomedical Sciences for Health (SCIBIS), 
University of Milan, Via G. Colombo 71, 20133 Milan, Italy
2 
IRCCS, Istituto Ortopedico Galeazzi, Via R. Galeazzi 4, 
20161 Milan, Italy
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Introduction
A successful aerobic performance depends on several 
physiological, biomechanical, and psychological factors 
(Bentley et al. 2007; Coyle 1995). Among physiological 
aspects, a high maximum pulmonary oxygen uptake ( ̇V
O2max), the ability to maintain a long time to exhaustion 
at V̇O2max, a faster ̇VO2-transition from rest to steady-
condition ( ̇VO2 kinetics), a higher lactate threshold and a 
low  O2 cost of running are the main parameters of aerobic 
performance (Poole and Richardson 1997; Coyle 1995; 
Poole and Jones 2012).
Also the maximum aerobic velocity  (Vmax), defined 
as the minimum velocity capable to elicit ̇VO2max when 
considering only the completion of the primary phase of 
̇VO2-on kinetics (Ferretti 2015), is reported as a strong 
marker of running performance (Bentley et al. 2007) and 
it integrates both metabolic and biomechanical aspects of 
running into a single factor (Buchheit and Laursen 2013). 
In elite aerobic athletes, a higher  Vmax reflects a greater 
capacity to utilize the aerobic metabolic pathways across 
several sports (Noakes 1988; Pedro et al. 2013; Ziogas 
et al. 2011; Rampinini et al. 2007).
̇VO2max and  Vmax are generally determined using dif-
ferent incremental running protocols (Kuipers et al. 2003; 
Riboli et al. 2017), among which continuous or discontinu-
ous tests that may vary in work rate increments and stage 
duration (Billat et al. 1996; Kuipers et al. 2003; Riboli 
et al. 2017). Discontinuous incremental protocols (DP) 
are characterized by constant work rates interspersed by 
resting periods (Duncan et al. 1997; Riboli et al. 2017). DP 
permits to reach an equilibrium between the cardiorespira-
tory and metabolic systems and the work rate when lasting 
at least three minutes to achieve a steady-state condition 
(Poole and Jones 2012). However, the long overall dura-
tion of DP would markedly lengthen the whole testing 
phase, thus affecting the possibility to test several athletes 
within one single session, as often required in sports prac-
tice. Conversely, incremental continuous protocols (CP) 
last short overall duration and they have been shown as a 
valid and reliable method to determine ̇VO2max despite the 
submaximal physiological adjustments cannot be reached 
as in DP due to increments in work rate faster than cardi-
orespiratory and metabolic adjustments (Riboli et al. 2017, 
2021). Despite in some intermittent protocols with very 
low workload vs recovery ratio ̇VO2max may not be reached 
(Vinetti et al. 2017), previous studies using CP and DP 
showed that ̇VO2max was found to be independent from 
the protocol adopted (Kuipers et al. 2003; Riboli et al. 
2017, 2021). Conversely, testing protocols with shorter 
stage duration may lead to higher  Vmax (Riboli et al. 2017; 
Kuipers et al. 2003; Adami et al. 2013). Given that  Vmax is 
currently utilized to prescribe or monitor training routines 
(Buchheit and Laursen 2013; Riboli et al. 2021), a precise 
 Vmax assessment may allow coaches to manipulate accu-
rately the physiological load during running exercises as 
a percentage of  Vmax (Buchheit and Laursen 2013; Riboli 
et al. 2021). For instance, 90–110% of  Vmax are suggested 
for long-interval exercises, 110–130%  Vmax for short-inter-
vals exercises, 130–160%  Vmax for repeated sprint train-
ing and > 160%  Vmax for sprint interval training (Buchheit 
and Laursen 2013). Therefore, a precise  Vmax assessment 
should be carefully taken into account for athletes’ test-
ing and training prescription (Riboli et al. 2017; Bentley 
et al. 2007).
Athletes with a high aerobic capacity (HI), such as long- 
and middle-distance runners, are qualified by greater physi-
ological characteristics in terms of high ̇VO2max and fast 
̇VO2 kinetics (Coyle 1995) than in individuals with lower 
aerobic capacity (LO). A high ̇VO2max represents, indeed, a 
pronounced maximal pulmonary, cardiovascular, metabolic 
and muscular capacity to uptake, transport and utilize  O2 
(Poole and Richardson 1997). Moreover, rapid ̇VO2 kinetics 
may lead to a smaller  O2 deficit and a reduced intracellu-
lar perturbation, thus reflecting greater exercise tolerance 
(Poole and Jones 2012; Dupont et al. 2005) and endurance 
performance (Poole and Jones 2012). These characteristics 
in HI may therefore lower or even minimize the misestimat-
ing issue that may occur in CP because of their faster ̇VO2 
kinetics.
With this in mind, the present study aimed to investi-
gate how aerobic training status may affect  Vmax assessment 
during CPs vs DP in two groups of athletes, characterized 
by different aerobic training conditions. Should HI in the 
investigated group demonstrate faster ̇VO2 kinetics due to 
their greater ability of the cardiorespiratory and metabolic 
systems to adjust to continuous increases in work rate typi-
cal of CP, the  Vmax misestimating issue may be minimized, 
when comparing their CPs to DP results.
Materials and methods
Participants
Sixteen well-trained middle and long-distance runners 
(age: 22.1 ± 1.8 years; stature: 1.75 ± 0.05 m; body mass: 
70.3.7 ± 3.7 kg; mean ± standard deviation) volunteered to 
participate in the study and were classified into two groups, 
according to their higher (HI) or lower (LO) ̇VO2max and the 
International Physical Activity Questionnaire (IPAQ). All 
participants met the following criteria: (a) more than four 
years of systematic training and (b) no injuries in the last 
year. The ethics committee of the local University approved 
the study (protocol #102/14) which was performed in 
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accordance with the principles of the Declaration of Hel-
sinki (1964 and updates). All participants gave their written 
consent after a full explanation of the purpose of the study 
and the experimental design.
Study design
To test the current hypothesis, two incremental continuous 
protocols with different stage durations (CP) were performed 
and compared to a discontinuous incremental protocol (DP). 
The present study spanned over a maximum of 3 weeks. The 
participants reported to the laboratory five times, separated 
by at least 72 h. During the first visit, they were familiar-
ized with the experimental procedures. During the second 
session, they performed a continuous incremental protocol 
(1 km⋅h−1 per minute) to determine ̇VO2max and to complete 
the IPAQ. Within the remaining three sessions, the partici-
pants randomly underwent the three experimental condi-
tions (two continuous and one discontinuous incremental 
protocols). Within each testing-session, an initial 5-min 
submaximal bout at 10 km⋅h−1 was modelled to determine 
the on-transient ̇VO2 kinetics. Participants were instructed to 
avoid any form of strenuous exercise in the three days before 
each session. In addition, they were asked to have their last 
standardized meal at least three hours before each session. 
Finally, they were requested to abstain from ergogenic and 
caffeinated beverages before testing.
Participants were split subsequently into two groups, 
according to their ̇VO2max normalized per body mass 
(ml·kg−1·min−1) and their training routines (i.e., n of train-
ing sessions per week). The first HI group was characterized 
by a higher ̇VO2max and more than five training sessions per 
week. The second LO group was characterized by a lower 
̇VO2max and no more than three training sessions per week.
Experimental procedures
All tests were conducted approximately at the same time 
of the day in a climate-controlled laboratory (constant tem-
perature of 20 ± 1 °C and relative humidity of 50 ± 5%). 
All tests were carried out on a treadmill ergometer (RAM 
s.r.l., mod. 770 S, Padova, Italy) with a 1% positive slope. 
Blood lactate concentration  (BLa−) was assessed by a 
spectrophotometric system (Lactate Pro LT-1710, Arkray, 
Kyoto, Japan). The lactate analyzer was calibrated before 
each protocol to guarantee consistent data. ̇VO2max, expira-
tory ventilation, carbon dioxide production and respiratory 
exchange ratio were measured during each protocol by a gas 
analyzer cart (Cosmed, mod. Quark b2, Rome, Italy). The 
device was calibrated before each test with gas mixtures of 
known concentration  (O2 16%,  CO2 5%, balance  N2). Heart 
rate was monitored continuously using a heart rate monitor 
(Polar Electro Oy, mod. S810i, Kempele, Finland). Arterial 
 O2 saturation was determined by a finger-tip infrared oxym-
eter (NONIN Medical, mod. 3011, Minneapolis, MN). At 
the end of the test, the rate of perceived exertion (RPE) was 
determined using the 6–20 Borg scale for general, respira-
tory and muscular fatigue. The participants were strongly 
encouraged by the operators to perform each test up to their 
maximum exercise capacity.
Continuous Incremental Protocol 1 (CP1). After 5 min 
of baseline measurements, while standing on the treadmill, 
the participants warmed up at 10 km⋅h−1 for 5 min. Then, 
the running speed was increased progressively by 1 km⋅h−1 
per minute until volitional exhaustion.  BLa− was meas-
ured at baseline, at the end of each stage and after 1, 3 and 
5 min of passive recovery. The achievement of V̇O2max was 
identified as the plateauing of ̇VO2 (< 2.1 ml·kg−1·min−1 
increase) despite an increase in workload (Poole and Rich-
ardson 1997). If the above-stated criterion and/or second-
ary criteria to establish ̇VO2max (Poole et al. 2008) were not 
fulfilled, the participants were asked to perform a further 
constant-speed test equal or higher than the highest speed 
achieved at the end of the incremental test, as strongly rec-
ommended (Rossiter et al. 2006). ̇VO2, carbon dioxide pro-
duction, expiratory ventilation,  O2 saturation and respiratory 
exchange ratio were averaged during the last 30 s of each 
step at submaximal workload and over the last 30 s before 
exhaustion.  Vmax was determined as the minimal running 
velocity that elicited ̇VO2max over a period of 30 s (Billat 
et al. 1996). If a stage could not be completed, the  Vmax 
was calculated according to a previously published equation 
(Kuipers et al. 2003)  [Vmax =  Vcompleted + t/T x speed incre-
ment], in which  Vcompleted is the running speed of the last 
stage that was completed, t the number of seconds that the 
uncompleted running stage could be sustained, T the number 
of seconds required to complete the stage, and speed incre-
ment is the speed load increment in km⋅h−1.
Continuous Incremental Protocol 2 (CP2). CP2 followed 
the same experimental procedures as CP1, but with the 
increases in treadmill running speed of 1 km⋅h−1 every two 
minutes. As for CP1, ̇VO2, carbon dioxide production, expir-
atory ventilation,  O2 saturation, and respiratory exchange 
ratio were averaged during the last 30 s of each step at sub-
maximal workload and over the last 30 s before exhaustion. 
 Vmax was determined as the minimal running velocity that 
elicited ̇VO2max over a period of 30 s (Billat et al. 1996).
Discontinuous Incremental Protocol (DP). DP protocol 
involved five workloads of 4 min each, interspersed by at 
least 5 min of recovery (Bernard et al. 2000). The optimal 
stage duration suggested for DPs is still questioned (Bernard 
et al. 2000). Although some authors suggested that it should 
be around 6–8 min (Bernard et al. 2000), it was criticized 
that relatively long stage duration could result in prema-
ture fatigue and suggested that 4–6 min could be suitable 
for this purpose (Bentley et al. 2007; Kuipers et al. 2003; 
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European Journal of Applied Physiology (2021) 121:3083–3093
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Bernard et al. 2000). Since shorter test duration is strongly 
advocated during in-field practice, a 4-min stage duration 
was used here.
Baseline measurements were recorded with the partici-
pants standing on the treadmill. The first two workloads were 
set at 8 and 10 km·h−1 for all participants. The following 
three workloads were tailored for each participant according 
to the individual cardiorespiratory responses to the first two 
workloads and considering the theoretical maximum heart-
rate determined (Bernard et al. 2000). Firstly, based on the 
̇VO2 and the heart-rate recorded during the first two stages, 
a sub-maximal linear regression was determined up to the 
predicted peak heart rate, to predict the speed correspond-
ing to possible exhaustion (Bernard et al. 2000). Then, the 
third, the fourth and the fifth workloads corresponded to 
approximately 80%, 90% and 105% of the predicted peak 
workload, respectively. The fourth and the fifth workloads 
were recalculated using the heart-rate and ̇VO2 recorded dur-
ing the third and the fourth stage, respectively. The last stage 
was tailored to let the participants maintain the task for at 
least four minutes (Bernard et al. 2000). The blood lactate 
concentration was measured at baseline and after 1, 3 and 
5 min of passive recovery for each workload, and the peak 
blood lactate was inserted into the data analysis. ̇VO2, car-
bon dioxide production, expiratory ventilation,  O2 saturation 
and respiratory exchange ratio were determined as the aver-
age value of the last (fourth) minute during each workload 
(Poole and Richardson 1997).  Vmax was extrapolated from 
the regression analysis equation of ̇VO2 as a function of run-
ning velocity at submaximal workloads below the lactate 
threshold (Bernard et al. 2000; Riboli et al. 2017).
Lactate threshold, ̇VO2/Velocity slope 
at submaximal exercise and ̇VO2 kinetics
Lactate threshold was determined by the  DMAX method, 
according to which it was identified as the point on the 
third-order polynomial curve that yielded the maximal per-
pendicular distance to the straight line formed by the two 
end data points (Riboli et al. 2019). Similar to the previous 
study, lactate threshold calculated from CP1 was utilized to 
limit the range of exercise during which the ̇VO2 vs running 
velocity relationship at submaximal exercise was considered 
(Riboli et al. 2017).
̇VO2/Velocity slope: the ̇VO2/Velocity slope was calcu-
lated as the regression analysis of the ̇VO2 vs velocity rela-
tionship at submaximal workloads below lactate threshold 
for CP1, CP2 and DP (Anderson 1996; Fletcher et al. 2009).
̇VO2 kinetics. The on-transient ̇VO2 kinetics were mod-
elled after four different bouts of 5-min submaximal exer-
cise (10 km·h−1, moderate intensity, below lactate thresh-
old) to avoid any effect of the slow component phenomenon 
(Jones et al. 2011). The influence of the inter-breath noise 
was reduced averaging the results of four identical tests in 
each participant (Lamarra et al. 1987). Each abnormal breath 
(e.g., different from the mean of the adjacent four data point 
by more than three times the standard-deviation of those 
four point, were excluded (Dupont et al. 2005). To increase 
the time resolution the breath-by-breath ̇VO2 data were sub-
sequently linearly interpolated, and the four data sets were 
averaged together to produce a single response for each sub-
ject. This procedure was previously established to reduce the 
noise of the ̇VO2 signal and to provide the highest confident 
results (Poole and Jones 2012). The on-transient of the ̇V
O2 kinetics were modelled as previously proposed (Barstow 
and Mole 1991). The time-delay of the cardiodynamic-phase 
and the time-constant of the primary-phase (i.e., the time 
to reach 63% of the ̇VO2 steady-state of the ̇VO2 kinetics 
were calculated to determine the amplitude of ̇VO2 from 
baseline to steady-state (Poole and Jones 2012). Then, the 
mean response time of the on-transition ̇VO2 kinetics as the 
sum of time-delay and time-constant was calculated. The 
time-delay, the time-constant and the mean response time 
were thereafter inserted into data analysis.
Statistical analysis
Statistical analysis was performed using a statistical software 
package (Sigma Plot for Windows, v 12.5, Systat Software 
Inc., San Jose, CA, USA). To check the normal distribution 
of the sampling, a Kolgomorov-Smirnov test was applied. A 
one-way analysis of variance (ANOVA) for repeated meas-
ures was used also to assess significant differences in  Vmax, 
̇VO2max, carbon dioxide production, respiratory exchange 
ratio, arterial  O2 saturation, heart-rate, expiratory ventila-
tion, blood lactate concentration, ̇VO2/Velocity slope (for 
both slope and intercept of the submaximal regression analy-
sis equation), ̇VO2 kinetics (time-delay, time-constant and 
mean-response time), general-, muscular-, and respiratory-
RPE between CP1, CP2 and DP. For all pairwise multiple 
comparisons, a post-hoc Shapiro–Wilk test was applied. 
A regression analysis was used to assess the relationship 
between ̇VO2 and running velocity at submaximal exercise. 
The magnitude of the changes was assessed using Cohen’s 
standardized effect size (ES) with 95% confidence inter-
vals (95% CI). Effect size with 95% CI was calculated and 
interpreted as follows: < 0.20: trivial; 0.20–0.59: small; 
0.60–1.19: moderate; 1.20–1.99: large; ≥ 2.00: very large 
(Hopkins et al. 2009). Pearson’s product moment and 95% 
CI were utilized to assess the relationship among protocols 
for  Vmax. The correlation coefficients were interpreted as 
follows: r < 0.1 trivial; 0.1 ≤ r < 0.3 small; 0.3 ≤ r < 0.5 mod-
erate; 0.5 ≤ r < 0.7 large; 0.7 ≤ r < 0.9 very large; 0.9 ≤ r < 1 
nearly perfect. Statistical significance was set at an α level 
of 0.05. Unless otherwise stated, all values are presented as 
mean ± standard deviation (SD).
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Results
Between‑groups differences
As shown in Table 1,  Vmax [P < 0.001, (ES:1.85/2.59)], 
̇VO2max [P < 0.001, (ES:0.85/1.07)],  VCO2 [P < 0.001, 
(ES:0.22/0.61)] and VE [P < 0.001, (ES:0.57/0.82] were 
small to very largely higher in HI than LO within-each 
protocol (CP1, CP2 and DP) (Table 1). No between-groups 
differences (P > 0.05) in respiratory exchange ratio, arte-
rial  O2 saturation, heart rate,  BLa−
peak, general-, respira-
tory-, and muscular-RPE were found.
The lactate threshold calculated in CP1 was moder-
ately [ES:1.99(CI:0.79/3.19)] higher (P < 0.001) in HI 
[17.8(1.1)] than LO [16.1(0.3)]. Overall, the submaxi-
mal regression analysis of ̇VO2/velocity relationship for 
CP1, CP2 and DP was less steep (P < 0.05) in HI than 
LO (Fig. 1); in details, the intercept of the submaximal 
regression analysis in ̇VO2/velocity relationship ( ̇VO2/
velocity intercept) was moderately to largely (ES:-0.86/-
1.63) lower (P < 0.05) in HI than LO within-each protocol 
(CP1, CP2 and DP). The slope of the submaximal regres-
sion analysis in ̇VO2/velocity relationship ( ̇VO2/velocity 
slope) showed trivial to moderate (ES:-0.18/0.83) not 
significant (P > 0.05) differences between HI and LO in 
CP1, CP2 and DP.
The ̇VO2 kinetics was largely to very largely (ES: 
-2.74/-1.76) faster (P > 0.05) in HI than LO: despite 
small [ES:-0.36(CI: -1.35/0.63] non-significant 
differences (P > 0.05) in time-delay, HIGH showed a 
large [ES:-1.76(CI:−2.92/−0.61] and very-large [ES:-
2.74(−4.10/−1.37)] difference with a faster time-constant 
and mean-response time than LO, respectively (Fig. 2).
Between‑protocols differences at maximal exercise
As shown in Table 1,  Vmax was largely higher in CP1 
vs DP for both HI [P < 0.001, ES:1.96(0.77/3.16)] and 
LO [P < 0.001, ES: 1.84(0.67/3.01)]. In CP1 vs CP2, 
 Vmax was largely higher for HI [P < 0.001, ES: 1.73, CI: 
0.58/2.88)] and moderately higher for LO [P = 0.006, 
ES: 1.11(0.06/2.17]. In CP2 vs DP,  Vmax was moderately 
higher for LO [P = 0.039, ES: 0.75(−0.26/1.76)], while 
small not significant  Vmax-difference for HI [P = 0.102, ES: 
0.30(−0.68/1.29)] were retrieved.
No between-protocol (CP1 vs CP2 vs DP) differences for 
maximum  ̇VO2,  VCO2, RER,  SaO2, fH, VE and  BLa−
peak 
were found for both HI and LO. Similarly, no between-pro-
tocol differences in general-, respiratory- and muscular-RPE 
were found.
Between‑protocols differences at submaximal 
exercise
As shown in Fig.  1,  ̇VO2/velocity slope showed a 
moderate difference in CP1 vs DP for HI [P = 0.003, 
ES:−0.85(−1.88/−0.17)] and a large difference for LO 
[P = 0.002, ES: −1.75(−2.91/−0.60)]. In CP1 vs CP2,  ̇VO2/
velocity slope showed a small difference for HI [P = 0.003, 
Table 1  Cardiorespiratory, metabolic, and perceptual variables at maximum exercise for HI and LO groups. Mean (SD)
Vmax velocity associated with maximum oxygen uptake; ̇VO2 oxygen uptake; ̇VCO2 carbon dioxide production; RER respiratory exchange ratio; 
SaO2 arterial  O2 saturation; fH heart rate frequency; ̇VE, expiratory ventilation; BLa−
peak peak blood lactate concentration; and rate of perceived 
exertion (RPE) at general, respiratory, and muscular level. Variables were determined at maximum exercise in the three testing conditions (CP1, 
continuous ramp 1; CP2, continuous ramp 2; DP, discontinuous protocol).
* P < 0.05 vs DP; **P < 0.05 vs CP1; ***P < 0.05 vs HI
HI
LO
CP1
CP2
DP
CP1
CP2
DP
Vmax (km·h−1)
22.1 (1.2)*
19.9 (1.2)*, **
19.5 (1.3)
19.1 (1.8)*, ***
17.2 (1.4) *,**,***
16.2 (1.1)***
̇VO2 (ml·min−1)
4169.6 (478.9)
4132.8 (134.2)
4158.8 (473.5)
3912.0 (442.6)***
3907.8 (356.4) ***
3895.3 (424.9) §
̇VO2 (ml·kg·min−1)
59.2 (5.2)
58.7 (5.4)
59.1 (5.2)
54.6 (4.8)***
54.4 (4.1) ***
54.5(2.5)***
̇VCO2 (ml·min−1)
4581.9 (510.4)
4492.8 (110.8)
4665.2 (442.0)
4465.8 (494.7)***
4366.4 (473.0) ***
4371.7 (463.0) ***
RER
1.10 (0.09)
1.09 (0.03)
1.13 (0.04)
1.13 (0.06)
1.11 (0.06)
1.12 (0.06)
SaO2 (%)
89.8 (2.7)
89.6 (1.8)
89.8 (2.7)
91.0 (1.7)
90.6 (2.7)
90.1 (2.7)
fH (beats·min−1)
188.0 (10.0)
188 (10.0)
186.0 (7.0)
189.0 (1.0)
188.0 (5.0)
187.0 (7.0)
̇V E (l·min−1)
166.9 (19.4)
164.1 (4.2)
163.3 (10.9)
155.1 (19.4)***
156.2 (14.9) ***
155.4 (7.0) ***
BLa−
peak (mM)
13.0 (4.0)
11.4 (2.3)
12.5 (2.1)
11.4 (1.3)
11.9 (1.0)
11.8 (0.8)
General RPE (au)
18.2 (1.2)
17.9 (1.3)
18.0 (1.3)
18.1 (2.1)
18.3 (1.5)
18.9 (1.2)
Respiratory RPE (au)
18.5 (1.2)
17.7 (1.4)
17.7 (1.4)
17.6 (3.1)
17.8 (1.7)
18.8 (1.0)
Muscular RPE (au)
17.4 (1.5)
17.9 (1.8)
18.4 (1.5)
17.8 (1.7)
17.9 (2.6)
18.1 (1.9)
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European Journal of Applied Physiology (2021) 121:3083–3093
1 3
ES:−.48(−1.48/0.51)] and very large difference for LO 
[P = 0.007, ES: -5.97(−8.26/-3.68)]. In CP2 vs DP,  ̇VO2/
velocity slope showed a trivial no-significant difference for 
HI [P = 0.283, ES: −0.20(−1.18/0.79)] and a very large dif-
ference for LO [P = 0.016, ES: −2.33(−3.60/−1.06)].
In CP1 vs DP,  ̇VO2/velocity intercept showed a small dif-
ference for HI [P < 0.001, ES:0.21(−0.78/1.19)] and a mod-
erate difference for LO [P = 0.002, ES: 0.99(0.05/2.03)]. In 
CP1 vs CP2,  ̇VO2/velocity intercept showed a trivial differ-
ence for HI [P = 0.010, ES:0.10(-0.88/1.08)] and a moderate 
difference for LO [P = 0.015, ES:0.61 (-0.36/1.60)]. In CP2 
vs DP,  ̇VO2/velocity intercept showed a trivial no-signifi-
cant differences for HI [P = 0.348, ES: 0.00(−0.98/0.98)] 
and a very large difference for LO [P < 0.001, ES: 
1.51(0.40/2.62)].
Between‑protocol Vmax correlations
Very large between-protocol correlations for  Vmax were cal-
culated for HI (r = 0.73, r = 0.84, and r = 0.73 for CP1 vs DP, 
CP2 vs DP and CP1 vs CP2, respectively P < 0.05). Mod-
erate to large between-protocol correlations for  Vmax were 
calculated for LO (r = 0.49, r = 0.68, and r = 0.79 for CP1 
vs DP, CP2 vs DP and CP1 vs CP2, respectively P < 0.05).
Relationship between training status 
and between‑protocol differences
The percentage of the  Vmax in CP1 vs DP showed a small 
[P = 0.045, ES: -0.53 (-1.56/0.46)] difference between HI 
and LO [+ 13.3(5.4)% and + 17.9(10.2)%,, respectively] and 
a moderate [P = 0.032, ES:−0.75 (−1.76/0.26)] difference 
for CP2 vs DP [+ 6.2(6.6) and + 2.1(3.7)% for HI and LO, 
respectively].
As shown in Fig. 3, the percentage of the  Vmax-difference 
in CP1 than DP showed an inversely large correlation with 
 Vmax,  ̇VO2max and the velocity at lactate threshold. Con-
versely, the percentage of the  Vmax-difference in CP1 than 
DP was largely correlated with the time-constant of the  ̇V
O2 kinetics.
Discussion
The main finding of the present study was that HI, with 
faster  ̇VO2 kinetics, had lower differences in  Vmax between 
CP and DP than LO. This observation may confirm the 
experimental hypothesis stating that athletes with higher 
aerobic capacity and faster  ̇VO2 kinetics are able to adjust 
better to work rate increments typical of CP with short stage 
duration. Noticeably, HI had a similar  Vmax in DP and CP2 
(i.e., the continuous protocol with slower work rate incre-
ments) and the difference in  Vmax between CP1 and DP was 
lower than in LO. Lastly, the percentage of the  Vmax differ-
ences between CP1 and DP were inversely correlated with 
 Vmax,  ̇VO2max and directly correlated to the time-constant of 
the  ̇VO2 kinetics, providing further evidence that between-
protocol  Vmax differences in HI are minimized likely because 
of their faster  ̇VO2 kinetics.
Fig. 1  The ̇VO2 as a function of running velocity at submaximal work 
rates (below the velocity corresponding to the lactate threshold cal-
culated in CP1 condition) for both HI and LO. The solid, dashed and 
dotted lines represent the regression lines for the discontinuous (DP), 
continuous protocol with 1 km·h−1 increment per minute (CP1) and 
2 km·h−1 increment every 2 min (CP2), respectively. Panel A and B 
show HIGH and LOW group, respectively. Regression equations 
(y = a · bx) and correlation coefficients are also reported. *P < 0.05 
vs DP for slope and intercept of the regression equation, #P < 0.05 vs 
CP1 for slope of the regression equation, §P < 0.05 vs HI for the inter-
cept of the regression equation
3089
European Journal of Applied Physiology (2021) 121:3083–3093 
1 3
Preliminary considerations
The present results came with no between-protocol differ-
ences in  ̇VO2max and in the other main cardiorespiratory 
and metabolic parameters in both HI and LO. Despite some 
previous findings about the effects of protocol (i.e. workload 
vs recovery ratio) on  ̇VO2max (Vinetti et al. 2017), these 
findings reinforce previous data demonstrating that  ̇VO2max 
was independent of the protocol adopted across different 
incremental testing procedures (Bentley et al. 2007; Billat 
et al. 1996; Riboli et al. 2017). The present outcomes are in 
line with previous literature, in which no differences in  ̇V
O2max were observed between protocols in different popu-
lations, such as recreationally-active men (Kirkeberg et al. 
2011), physically-active young adults (Riboli et al. 2017), 
semi-professional soccer players (Riboli et al. 2021) and 
competitive middle- and long-distance runners (Billat et al. 
1996; Kuipers et al. 2003). Similar results were also found 
in moderately-active cyclists during cycle-ergometric evalu-
ation (Adami et al. 2013).
Maximum exercise
The present findings demonstrate that  Vmax was protocol-
dependent, as also previously observed (Kuipers et al. 2003; 
Riboli et al. 2017, 2021). The steeper the work rate increase, 
the higher the  Vmax in both groups. In LO  Vmax differed 
in each protocol (i.e., CP1 > CP2 > DP). Conversely, in HI 
the  Vmax differences between CP2 and DP were not present 
(i.e., CP1 > CP2 = DP). These findings suggest that higher 
aerobic capacity may minimize the between-protocol  Vmax 
Fig. 2  The rate of  ̇V O2 increase at submaximal exercise for both 
HI and LO. Panel A shows the rate of  ̇VO2 increase ( ̇VO2 kinetic) 
for two representative subjects (HI: white circles; LO: black circles). 
The time-delay (Panel B), the time-constant (Panel C) and the mean-
response time (Panel D) are illustrated for each subject (white circles) 
in HI (white bar) and LO (dark-grey bar) group. #P < 0.05 vs HI
3090
 
European Journal of Applied Physiology (2021) 121:3083–3093
1 3
differences due to the faster cardiorespiratory and metabolic 
adjustments to match the increasing mechanical power in 
CP. This explanation was further supported by the faster 
̇VO2 kinetics in HI, in which no difference was found 
between CP2 and DP. On the contrary, in LO  Vmax in CP2 
was higher than in DP due to the slower  ̇VO2 kinetics. A 
direct comparison with previous studies is challenging, as 
this was the first study investigating the effect of aerobic 
training status on  Vmax. Previous studies observed a greater 
between-protocol difference when steeper work rate vs time 
increments were utilized (Kuipers et al. 2003; Riboli et al. 
2017, 2021). Indeed, when comparing three CPs with 1-, 
3- or 6-min stage duration in competitive middle-distance 
runners,  Vmax was related to the slope of the work rate vs 
velocity increments (Kuipers et al. 2003). Similar results 
were found when a CP with different work rate vs veloc-
ity increments was used during cycle ergometry in active 
people (Adami et al. 2013) or international competitive tri-
athletes (Bentley and McNaughton 2003). Recently, greater 
peak mechanical power output was found also in healthy 
participants using a synchronous arm crank ergometry when 
work rate increments were steeper (Kouwijzer et al. 2019). 
Interestingly, when long-distance runners were tested using 
CP with different stage duration but similar slope in the 
velocity vs time increments (e.g., 1 km·h−1 increments every 
2 min vs 0.5 km·h−1 increments every min), no difference in 
 Vmax was detected (Billat et al. 1996). Similar findings were 
observed also in sedentary men on cycle ergometer (Zhang 
et al. 1991).
Submaximal exercise
A faster  ̇V02 kinetics was observed in HI than in LO partici-
pants during the test at 10 km/h, implying a more rapid car-
diorespiratory and metabolic adjustment capacity to match 
mechanical power increase during incremental exercise. 
Previous investigations observed that athletes with a high 
aerobic capacity, such as long- and middle-distance run-
ners, were qualified by greater physiological characteristics 
in terms of faster  ̇VO2 kinetics (Poole and Jones 2012; Coyle 
1995). In top-level aerobic athletes, indeed, an extremely 
short time (i.e., ~ 30 to ~ 40 s) is required to achieve a  ̇VO2 
steady-state (Poole and Jones 2012), while in trained healthy 
individuals at least 2–3 min or even more are required (Rob-
ergs 2014; Poole and Jones 2012). The present results con-
firm the current hypothesis demonstrating a lower between-
protocol  Vmax difference in HI than in LO likely due to the 
changes in running velocity faster than cardiorespiratory and 
metabolic adjustments. This was remarkably highlighted by 
no-differences in  Vmax between CP2 and DP for HI.
The between-protocol difference in the  ̇VO2/velocity 
slope, was greater in LO (large to very large) than in HI 
(trivial to moderate), leading the slope to CP1 > CP2 > DP 
Fig. 3  Relationship between training status and the between-protocol  Vmax dif-
ference. The percentage of the individual  Vmax-difference in CP1 than DP is 
related with the velocity associated with maximum oxygen uptake  (Vmax, Panel 
A), maximum oxygen uptake ( ̇VO2max, Panel B) and lactate threshold (LaT, 
Panel C). Regression equations (y = a · bx), 95% confidence intervals and cor-
relation coefficients are also reported
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European Journal of Applied Physiology (2021) 121:3083–3093 
1 3
and CP1 > CP2 = DP in LO and HI, respectively. High-level 
aerobic athletes are also qualified by better biomechanical 
characteristics matching with a faster  ̇V O kinetics and a 
higher running economy (Coyle 1995). In the present study, 
LO showed a reduced  ̇VO2/velocity slope in both CP1 and 
CP2 than DP, while in HI the difference between CP2 and 
DP disappeared. This condition typically occurs when the 
time to reach cardiorespiratory and metabolic equilibrium 
matches the change in work rate across CPs.
Training status and between‑protocol differences
The between-protocol  Vmax differences were inversely cor-
related with training status. A higher  ̇VO2max,  Vmax, lactate 
threshold and faster  ̇VO2 kinetics provided further evidence 
that between-protocol  Vmax differences in HI may be likely 
counteracted by their higher aerobic training status. There-
fore, a more consistent  Vmax across different protocols in 
athletes with a higher aerobic capacity was found. The 
knowledge of the between-protocols  Vmax differences could 
have practical implications for testing, exercise prescriptions 
and physiological outcomes during running activities. Dif-
ferent %  Vmax were shown to lead different physiological 
responses by increasing or decreasing the time spent at ~  ̇V
O2max, a crucial factor for chronic adaptations and perfor-
mance development (Buchheit and Laursen 2013). There-
fore, a more consistent  Vmax determination should permit a 
more accurate running exercise prescription in both HI and 
LO athletes.
Methodological considerations
Some methodological considerations should accompany 
the present investigation. First, the study of the dynamic 
response of metabolic and pulmonary variables upon exer-
cise onset is strongly affected by the recording technique 
(Ferretti 2015). The Auchincloss algorithm (Auchincloss 
et al. 1966) utilized to calculate dynamic  ̇VO2 responses 
requires a correct determination of the change in the amount 
of gas stored in the lungs over each breath. However, the 
algorithm estimated the end-expiratory lung volume impos-
ing fixed pre-defined values of end-expiratory lung volumes 
(Ferretti 2015) leading to an impossibility of attaining a cor-
rect estimation (di Prampero and Lafortuna 1989). Subse-
quently, it was demonstrated a two-time improvement of the 
signal-to-noise ratio in breath-by-breath alveolar gas transfer 
(Capelli et al. 2001) and a lower dynamic response (Cautero 
et al. 2002) using Grønlund algorithm. However, despite 
such algorithm improvements, the aforementioned issue 
could not be fixed (Ferretti 2015). Secondly, despite a step-
wise interpolation procedure was proposed to improve the 
time-constant calculation (Lamarra et al. 1987), a slightly 
higher time-constant than the interpolation interval still 
remains. Therefore, at least in the light exercise domain, 
mere stacking of multiple repetitions was proposed if the 
data were from the same  ̇VO2 on rest-to-exercise transient 
(Bringard et al. 2014; Francescato et al. 2014b, a). As such, 
attempts at improving the time resolution beyond the single-
breath duration could rely only on computational manipula-
tions, such as superimposition of several trials and interpola-
tion procedures (Francescato et al. 2014a; Francescato and 
Cettolo 2020).
Lastly, the present findings open to new future perspec-
tives. During submaximal running bouts, the time shift 
between velocity and  ̇VO2 could be calculated knowing the 
time constant of the  ̇VO2-on kinetics. Therefore, a mathe-
matical modeling would possibly provide a calibration equa-
tion for  Vmax correction in CP1 and CP2 with respect to DP.
Practical considerations
The between-protocol  Vmax differences in CP1 (+ 18% 
and + 13% than DP in LO and HI, respectively) and CP2 
(+ 6% than DP in LO) should be considered for both athletes 
aerobic profiling and exercise prescription. These results 
suggest that in LO a protocol with more than 2 min stage 
durations is required for the metabolic power to match the 
mechanical power. In HI, a 2-min stage duration may be 
suitable and can be consistently utilized within sport con-
texts. When shorter stage durations are mandatorily required 
(e.g., 1-min), a misestimate  Vmax should be considered to 
plan accurately high-intensity exercises in both HI and LO. 
Indeed, different %-  Vmax are suggested to increase the time 
spent at ~  ̇VO2max during high-intensity interval or intermit-
tent exercises (e.g., 110% to 130%-Vmax for short-intervals 
exercises or 130% to 160%-Vmax for repeated sprint train-
ings) (Buchheit and Laursen 2013). Therefore, when short 
intervals exercises (e.g., ~ 110%  Vmax) are prescribed, ~ 18% 
of  Vmax difference in CP1 vs DP for LO should induce an 
unexpected greater anaerobic involvement leading to acute 
physiological responses similar to a running exercise 
at ~ 130%-Vmax (i.e., ~ 25 km·h−1 instead of ~ 21 km·h−1). 
Similar differences between desired and actual physiologi-
cal responses could be found across any %-Vmax within both 
longer and shorter running exercises. Neglected between-
protocol  Vmax differences may mislead acute physiological 
responses (e.g., more aerobic or anaerobic contribution) and 
possibly negatively affect the training adaptations, especially 
within-athletes with lower training status. Therefore, the 
knowledge of the between-protocol differences may help 
practitioners to properly manage different testing modali-
ties and to adjust the %-Vmax when intermittent or interval 
running-based exercises are prescribed.
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European Journal of Applied Physiology (2021) 121:3083–3093
1 3
Conclusions
As previously observed, CP and DP can be used inter-
changeably to assess  ̇VO2max, but not  Vmax (Riboli et al. 
2017, 2021). We demonstrate here that aerobic training 
status can influence the magnitude of the between-protocol 
differences in  Vmax assessment. When different protocols 
are utilized to determine  Vmax, between-protocol differ-
ences exist, especially in CPs vs DP in which a matching 
between metabolic and mechanical power clearly occurs. 
These  Vmax differences should be considered when ath-
letes with different aerobic training status are tested. The 
 Vmax difference between CPs and DP disappeared in HI 
during CP2, suggesting that a protocol with at least 2-min 
stage duration may be sensitive enough in athletes with 
a greater aerobic capacity, while differences still exist 
across participants with lower aerobic training status for 
which at least 3-min stage duration seems required. These 
between-protocol  Vmax differences should be considered 
when athletes with different aerobic capacity are tested 
because they may affect the testing outcomes and training 
prescriptions.
Acknowledgements The authors would like to thank all the partici-
pants for their commitment.
Author contribution All authors contributed to the study. Conceptu-
alization: AR, FE, Data collection: AR, SR, EL, MB, EC, GC. Data 
analysis: AR, SR. Methodology: AR, FE. Visualization: AR, GC. Writ-
ing – original draft: AR, FE. Writing – review & editing: AR, FE.
Funding Open access funding provided by Università degli Studi di 
Milano within the CRUI-CARE Agreement. The authors have no fund-
ing supports to declare.
Availability of data and material Data and materials are available on 
request to the corresponding author.
Code availability Protocol #102/14.
Declarations 
Conflict of interest The authors have no conflicts of interest/competi-
tive interests to declare.
Ethical approval The ethics committee of the local University approved 
the study (protocol #102/14) which was performed in accordance with 
the principles of the Declaration of Helsinki (1964 and updates).
Consent to participate All participants gave their written consent after 
a full explanation of the purpose of the study and the experimental 
design.
Consent for publication All Authors give their consensus for publica-
tion.
Open Access This article is licensed under a Creative Commons 
Attribution 4.0 International License, which permits use, sharing, 
adaptation, distribution and reproduction in any medium or format, 
as long as you give appropriate credit to the original author(s) and the 
source, provide a link to the Creative Commons licence, and indicate 
if changes were made. The images or other third party material in this 
article are included in the article’s Creative Commons licence, unless 
indicated otherwise in a credit line to the material. If material is not 
included in the article’s Creative Commons licence and your intended 
use is not permitted by statutory regulation or exceeds the permitted 
use, you will need to obtain permission directly from the copyright 
holder. To view a copy of this licence, visit http:// creat iveco mmons. 
org/ licen ses/ by/4. 0/.
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Training status affects between-protocols differences in the assessment of maximal aerobic velocity.
Riboli, Andrea,Rampichini, Susanna,Cè, Emiliano,Limonta, Eloisa,Borrelli, Marta,Coratella, Giuseppe,Esposito, Fabio