Ergoline

An incremental ramp was performed on a mechanically braked cycle (Ergoselect 4 SN, Ergoline GmbG, Bitz, Germany) with cadence kept between 60–80 rpm. The protocol consisted of a three-minute initial workload of 50 Watts followed by a 1 watt increase every 3.6 s (equivalent to 50 Watts/3 min) until the volunteer’s voluntary exhaustion. Recordings of heart rate (HR) and RR intervals were taken continuously during the exercise test, as well as prior (PRE; after a period of habituation and session preparation) and post (POST) exercise by means of 3-min supine rest condition measurement intervals with two devices at the same time: (1) 12-channel ECG CardioPart 12 Blue (AMEDTEC Medizintechnik Aue GmbH, Germany; sampling rate: 500 Hz; desktop software: AMEDTEC ECGpro version 5.10.002), and (2) Polar H10 sensor chest strap device (Polar Electro Oy, Kempele, Finland; sampling rate: 1000 Hz; app software: Elite HRV App, Version 5.5.1). Placement of the ECG electrodes and chest strap device is pictured in Figure 2.
Breath-by-breath pulmonary gas exchanges were recorded throughout the ramp using a metabolic analyzer (Quark CPET, module A-67-100-02, COSMED Deutschland GmbH, Fridolfing, Germany; desktop software: Omnia version 1.6.5). Prior to testing, the gas analyzers were calibrated according to the manufacturer’s instructions. The protocol was terminated when the participant fell below a cadence of 60 rpm or due to self-determination. The following criteria served for the assumption of exhaustion: (A) heart rate >90% of the maximum predicted heart rate (prediction model according to [20 (link)]: 208 − (0.7 × age) and (B) respiratory quotient > 1.1. Maximum oxygen uptake (VO_2max) and maximum HR (HR_max) were defined as the average VO₂ and HR over the last 30 s of the test.

Body stature (cm) and body mass (kg) were measured, without shoes. Subjects performed the validation exercise protocol after performing a 5 min warmup (mild pedaling at 50 W) on a stationary cycling ergometer (Ergoline 800 s, Bitz, Germany) and a five min stretching. The Ergoline ergometer was selected, as it maintains steady power production in case that changes in revolutions per min (rpm) are detected. Thus, it was ensured that a constant load was applied at all times. The validation protocol was performed at a constant rate of 60–65 rpm during four exercise intensities (four stages). Each stage lasted for five min, starting from 75W and increasing by 25 W or 50 W according to the subjects perceived fitness, as estimated by the visual inspection of the heart rate response and the Borg Scale score. The intensity levels were classified to reflect relatively light, moderate and vigorous cardiorespiratory demands, according to the American College of Sports Medicine (ACSM) (Garber et al., 2011 (link)). Similar exercise intensity range has been previously selected by others (Rosdahl et al., 2010 (link); Perez-Suarez et al., 2018 (link)). Respiratory variables of VO₂, VCO₂, RQ and VE were calculated using values from the final minute of each five-min stage. During this timeframe, heart rate coefficient of variation (CV) was measured to be < 5%. Additionally, five 10-s averages were taken for these respiratory values and a CV below 10% was confirmed.
Measurements were made in-line (sequential gas sampling) with the COSMED - Quark CPET (Quark CPET) and PNOE (ENDO Medical, Palo Alto, CA) (Figure 1). The Quark CPET system acted as a reference standard, as it has been shown to be a valid metabolic system in its breath by breath mode (Nieman et al., 2013 (link)). The dead space between the two flow sensors has been quantified and accounted for in the calculations. The sequential in-line set up may provide advantages over repetitive assessment trials on different days since it eliminates the subject's biological day to day variation. The in-line connection of the two systems (Quark CPET and PNOE) was tested at a metabolic simulator (Relitech, Netherlands) prior to this validation study. A metabolic simulator was chosen for this test as it can generate fully reproducible conditions in terms of VE (Rosdahl et al., 2010 (link)). These tests were conducted to make sure that the in-line connection of the flow sensors (from the Quark CPET system and the PNOE system) does not affect ventilation (VE). In particular, the turbine of the Quark CPET system was connected with an adaptor to the MEMS (micro electro mechanical systems) based hot-film anemometer flow sensor of the PNOE system and the setup was tested at the metabolic simulator, in three different settings of simulated breathing frequency (BF = 20, 40, and 60 strokes per minute). VE in the metabolic simulator was set at 1 L throughout the experiments.
Once the in-line connection was tested, a second experiment was conducted with exactly the same procedure (BF = 20, 40, 60 strokes per minute), by mounting the turbine flow sensor of Quark CPET system to the metabolic simulator. The relative error between the in-line connection of the two flow sensors and the turbine of the Quark CPET system was 1.18%. The error of 1.18% lies within the technical specifications of the turbine flow sensor, as reported by the manufacturer (Cosmed, Rome), which is 1.5% when measuring VE.
The same procedure was followed with the PNOE system. VE measured by its standalone flow sensor was compared to the VE obtained from the in-line system. The relative error between the two measurements was 1.15%. Again, this error lies within the technical specification reported by the manufacturer of the PNOE system (ENDO Medical, Palo Alto, CA). These results demonstrate that the in-line connection of the two systems does not affect their operation and it can be used in this validation study. Such protocols have been successfully tested in the past (Prieur et al., 2003 (link)). Calibration of flow was performed with the two systems in line, using multiple pumps of a 3 L calibration syringe. Both devices were calibrated under specific gas concentrations after each measurement.
The reliability of the PNOE device was assessed by measuring gas exchange variables on ten randomly selected participants, on a separate day, at least 2 days after the first measurement. During this second visit, subjects were requested to adhere to similar nutritional habits before the repetition exercise protocol. The same experimental setup was followed in both visits.

Participants performed all the experimental trials on the same cycle ergometer (Ergoselect 200, Ergoline, Germany). Immediately following a standardized warm-up of 10 min at 50 W, all participants performed a ramp protocol with increments of 25 W·min^-1 until exhaustion. During GXT participants were monitored by standard 12 lead ECG (Quark T12, Cosmed, Italy), Oxygen consumption ( $\dot{\text{V}}{\text{O}}_2$ ) and carbon dioxide production ( $\dot{\text{V}}\text{C}{\text{O}}_2$ ) were recorded using breath-by-breath indirect calorimetry (Quark B², Cosmed, Italy). Familiarization GXT fulfilled three objectives: a) discard cardiac defects or diseases in any of the participants, b) to minimize the bias of progressive learning on test reliability and c) to discard any participant $\dot{\text{V}}{\text{O}}_{2\text{max}}$ lower than 55.0 ml·kg^-1·min^-1.
Both experimental maximal GXT with 15 min warm-up divided in three 5-min steady state stages at 45%, 55%, and 65% of the peak power output (PPO), being the three intensities below the second ventilatory threshold (VT₂). After 10 minutes of passive recovery in which each participant ingested 200–250 ml of water to ensure adequate hydration status, a sample of capillary blood from the finger was obtained to assess CBL (Lactate Pro, Arkray, Japan). Following, participants performed the GXT according to a modification of the protocol described by [4 (link)]. Initial workload was set at 50 W, with increments of 25 W·min^-1, requiring at all times a cadence between 80–85 rpm.
Heart rate was continuously monitored (RS400, Polar, Finland), gas exchange was recorded breath by breath using indirect calorimetry and capillary blood samples were obtained and analyzed every 2 min (i.e., each 50 W increments). Each participant indicated their rate of perceived exertion every two minutes using the Borg Scale 6–20, where 6 is defined as an effort "very very light" and one 20 "Maximum, strenuous" effort [18 ]. Capillary blood lactate analyzer and indirect calorimetry devices were calibrated before each test. In order to avoid the local acidosis that could impair the attainment of maximum cardiorespiratory performance, and according to the subjects’ maximal PPO in the GXT_PRE (i.e., 375-425W), starting at 50 W, the workload was progressively increased by 25 W·min^-1 that ensure that testing duration was not excessively long (i.e., 13.5–15.0 min). This protocol also allowed collecting between 7 to 9 capillary blood samples before exhaustion to be used in the CBL data analysis.

All participants underwent a graded CPET on a bicycle ergometer (Ergoselect 150P, ergoline GmbH, Bitz, Germany) within 1 week before HIIT. Minute ventilation ( $V$ _E) and oxygen consumption ( $V$ O₂) were measured breath by breath using a computer-based system (CareFusion MasterScreen CPX, CPX International Inc., Germany). $V$ O_2peak was defined as described in the ACSM guidelines for graded CPETs [26 ]. The  oxygen uptake efficiency sloe (OUES) during exercise was determined as described in our previous work [7 (link)]. A noninvasive continuous cardiac output (CO) monitoring system (NICOM, Cheetah Medical, Wilmington, DE, USA) was used to measure peak CO (CO_ex) during CPET. The CPET procedure and determination of cardiorespiratory parameters are detailed in Additional file 2.

We followed the previous protocol [7 (link)] for the hospital-based HIIT program using a bicycle ergometer (Ergoselect 150P, ergoline GmbH, Germany). Briefly, participants exercised at alternating intensities of 3-min intervals of 80% $V$ O_2peak and 3-min intervals of 40% $V$ O_2peak for 30 min in each session. They were instructed to complete 36 sessions of exercise training with a frequency of 2 to 3 sessions per week.