In recent years, there has been increased interest in fat metabolism in the field of both sports sciences and medicine. During long-distance events, increasing the reliance on fat can help athletes save glycogen reserves for high-intensity situations later in the events. The impairment of fat oxidation has been associated with the development of obesity and type 2 diabetes, and endurance training has been reported to improve fat oxidation and reduce body weight (Rosenkilde et al., 2015). With the aim of utilizing as much fat as possible in a certain period of time, the main strategy should be to exercise with low-moderate intensity. In particular, professional organizations such as the American College of Sports Medicine and American Medical Association recommend regular aerobic activity as part of a weight loss program with a special emphasis on low-intensity exercises for individuals interested in weight loss or control (Haskell et al., 2007).
It is well known that energy production shifts from fat to carbohydrates with an increase in the intensity of physical activity (Christensen and Hansen, 1939; Maunder et al., 2018). The specific intensity at which the fat oxidation rate is maximal (commonly presented as a percentage of the maximal oxygen uptake) is defined as Lipoxmax, Fatoxmax, or Fatmax by different researchers (Brun et al., 2011) and provides a measure of the maximal fat oxidation (MFO; the highest rate of fat oxidation observed at various intensities) (Randell et al., 2017). The determination protocol is often called the Fatmax test; exercise intensity could be individualized with the Fatmax test, and a suitable exercise prescription could be prepared according to the subjects' metabolic responses (Besnier et al., 2015; Dumortier et al., 2003; Tan et al., 2016). Athletic training programs, programs for weight or body fat loss, and exercise programs that treat or prevent welfare diseases may be the potential applications of Fatmax (Jeukendrup and Achten, 2001).
Lipids are oxidized predominantly at submaximal exercise intensities (
Depending on the approach of researchers, a specific time period of the Fatmax stage data is typically used to calculate substrate oxidation. In the studies in which 6 min intervals were chosen, investigators have used the last 30 s (Schwindling et al., 2014), 1 min (Isacco et al., 2015), 2 min (Besnier et al., 2015; Bordenave et al., 2008; Borel et al., 2015), or 3 min (Gmada et al., 2012; Marzouki et al., 2014) average values to calculate substrate oxidation. Although Achten et al. reported that 3 min intervals are appropriate for well-trained subjects to reach steady state (Achten et al., 2002), interval durations longer than 3 min have been recommended for sedentary individuals to avoid overestimating the fat oxidation rate (Bordenave et al., 2007). Furthermore, 3 min steps in the graded exercise protocol may be too short for obese individuals to reach steady state (Dandanell et al., 2017).
To our knowledge, changes in fat oxidation rates throughout the Fatmax stage have not been evaluated. Therefore, this study aimed to compare the MFO rates obtained from the stage average of Fatmax, last 2 min average of Fatmax, and highest value of Fatmax determined with a 6 min step protocol.
Participants and intervention content
A total of 35 healthy, sedentary males with an average age of 25.4 [+ or -] 0.7 years participated in this study (see Table 1 for anthropometric and physical characteristics). The study was explained to all participants in detail, and informed consent forms were acquired. Measurements were performed following the approval of the Ethics Committee and carried out in accordance with the Declaration of Helsinki. All tests were conducted at the Sports Physiology Research and Analysis Laboratory of the Physiology Department of Cukurova University, Medical Faculty. Participants with a history of any disease or drug use were excluded from the study. Calorie restrictions were not applied in terms of nutrition.
The participants visited the laboratory after 12 h of overnight fasting. Anthropometric measurements were performed before the exercise by the same person. Body mass and height were determined with a scale and a stadiometer. Circumference measurements were performed with a nonelastic measuring tape. Body fat estimates were derived according to Siri (1961). Body density, which was used in the Siri formula, was calculated for the men (Jackson and Pollock, 1978). The Martin formula was used to estimate body muscle mass (Martin et al., 1990).
Two separate exercise tests were performed at least 48 h apart. A maximal cardiopulmonary exercise test was performed on the first visit, and the Fatmax test was performed on the second visit. Both tests were performed on a treadmill (HP Cosmos, Nussdorf--Traunstein, Germany). Breath-by-breath gas measurements were taken throughout the exercise using an indirect calorimetric system (PFT Cosmed, Rome, Italy). The volume and gas calibration of the system was performed using a 3 L calibration syringe and calibration gases, respectively (16% [O.sub.2] and 5% C[O.sub.2]). The heart rate was recorded continuously by telemetry using a heart rate monitor (Cosmed, Rome, Italy).
Maximal cardiopulmonary exercise test
The participants started the test at 4 km/h, and the speed was increased by 0.5 km/h every min until exhaustion. The test was terminated if one or more of the following criteria were fulfilled: reaching up to 90% of the maximum heart rate according to the 220-age formula, formation of an oxygen uptake plateau, continuation of a non-protein respiratory quotient (npRQ) value at and over 1.15 (Balady et al., 2010).
Fatmax was determined with an incremental treadmill walking test after at least 12 h of fasting. The participants performed a 2 min warm-up at 3 km/h and started the test at 4 km/h. The speed was increased by 1.0 km/h every 6 min until the fat oxidation level was decreased to 0 with a npRQ value of 1.01 (Achten et al., 2002). The interval duration in the Fatmax test was selected based on a previous study (Perez-Martin et al., 2001).
Calculation of fat and carbohydrate oxidation
Breath-by-breath data were averaged in 10 s increments for the maximal cardiopulmonary test, and 60 s average values were used to calculate substrate oxidation in the Fatmax test. Substrate oxidation was calculated using the deviation from the stoichiometric equation (Frayn, 1983). Protein oxidation throughout the test was accepted as negligible. Fat and carbohydrate oxidation rates were calculated as follows:
Fat oxidation rate (g/min) = 1.67 x [??][O.sub.2] - 1.67 x [??]C[O.sub.2]
Carbohydrate oxidation rate (g/min) = 4.55 x [??]C[O.sub.2] - 3.21 x [??][O.sub.2]
By using the last 2 min average data, fat and carbohydrate oxidation rates were calculated for each stage, and the stage with the highest level of fat oxidation was recognized as Fatmax. In addition, the MFO rates were calculated with three different approaches in the Fatmax stage: (1) highest level of fat oxidation in the Fatmax stage (individual MFO rate), (2) 6 min average of fat oxidation in the Fatmax stage, and (3) last 2 min average of fat oxidation in the Fatmax stage. MacRae et al. (1995) proposed reference values to define the steady state level of gas exchange variables. In their study, during the 6 min progressive exercise, [??][O.sub.2], [??]C[O.sub.2], and VE were measured for the 5th and 6th min. In this period, [??][O.sub.2] and [??]C[O.sub.2] varied by less than 0.1 L/min...