Exercise is a fundamental way to oxidize lipids in order to prevent or mitigate metabolic syndrome. In recent years, maximal fat oxidation (MFO; i.e., the maximum value of fat oxidation) and [Fat.sub.max]([Fat.sub.max]=workload where greatest % of fat is oxidized to an incremental exercise load) have been investigated in a number of studies (Achten and Jeukendrup, 2003b; Sakamoto et al., 2012; Venables et al., 2005). It has been reported that MFO and [Fat.sub.max] are both below the ventilator threshold or approximately the same as the lactate threshold (Jeukendrup, 2004; Venables et al., 2005). Numerous factors such as sex, age, body and muscle composition, diet, training habits, physical activity, and lipid oxidation-related enzyme activity of skeletal muscle, all influence lipid metabolism and possibly affect fat utilization (Achten and Jeukendrup, 2003a; Brandou et al., 2006; Nordby et al., 2006; Stisen et al., 2006; Venables et al., 2005). [Fat.sub.max] can also be represented at the exercise intensity before carbohydrate % increases which is different than the workload shown by MFO. Exercise at [Fat.sub.max] is thought of as the intensity for the most useful workload for weight loss and lipid metabolism during training (Jeukendrup and Achten, 2001; Sakamoto et al., 2012; Venables et al., 2005). However, [Fat.sub.max] determined with the incremental exercise test is not necessarily the exercise intensity at which maximum percentage of lipid oxidation is attained during prolonged exercise. In fact, a previous study that compared lipid oxidation during prolonged exercise above, below, and at [Fat.sub.max] found no differences in lipid oxidation between exercise intensities, suggesting that [Fat.sub.max] may not be the optimal exercise intensity for maximal lipid oxidation (Schwindling et al., 2014).
Exercise intensity and duration also determine energy consumption. Exercise performed for the same duration at different intensities results in more energy consumption during the higher-intensity segments. A previous study reported increased lipid oxidation at +10%V[O.sub.2peak] than that at [Fat.sub.max] (Takagi et al., 2014). However, the exercise duration in the previous study was constant; it is likely that more energy was consumed during +10%V[O.sub.2peak] than at [Fat.sub.max]; thus, the study may have overestimated lipid oxidation during exercise. Additional research with controlled energy consumption during exercise is necessary to assess lipid oxidation at different exercise intensities.
Exercise intensity affects the secretion of hormones associated with energy substrate oxidation (Hansen et al., 2012; Van Loon et al., 2001; Wideman et al., 2002; Zouhal et al., 2008). Secretion of catecholamines that promote lipolysis, increases to a greater extent at higher relative exercise intensities; however, catecholamine secretion is not always increased at [Fat.sub.max] because exercise intensity is typically relatively low at this point (Hansen et al., 2012; Mohebbi et al., 2015; Van Loon et al., 2001; Zouhal et al., 2008). This does not mean that all lipids in blood, which are increased by hormones, are oxidized; rather, the amount of lipid oxidation varies according to relative exercise intensity (Klein et al., 1994; Romijn et al., 1993). Previous studies reported that carbohydrates are the main energy substrate in high-intensity exercise, compared with lipids being utilized more in lowto-moderate intensity exercise with the relative amount of lipids peaking at MFO and decreases during moderate intensity exercise (Romijn et al., 1993; Van Loon et al., 2001). Nonetheless, "moderate or lower intensity" represents a broad range of values, and it is possible that the substrate metabolism response also varies according to the exercise intensity within this range (Horowitz and Klein, 2000). A previous study has shown that the proportion of lipids used as the energy substrate in exercise significantly decreases above 60%V[O.sub.2max] (Friedlander et al., 1999) and they noted that fat oxidation rate decreased and switched more to carbohydrate. Therefore, in the present study, we comparatively examined exercise at [Fat.sub.max], at which the highest proportion of lipids is utilized, and at 60%V[O.sub.2max], at which the utilization of lipids significantly decreases.
It is possible that the levels of lipid oxidation are higher during exercise performed in the evening than the levels during exercise performed in the morning. A previous study on lipid oxidation at [Fat.sub.max] determined by using an incremental exercise test reported significantly higher oxidation levels in the evening than in the morning (Mohebbi and Azizi, 2011). However, it is unclear if lipid oxidation is greater for exercise performed at [Fat.sub.max] in the evening than the levels for exercise performed in the morning. Therefore, it is important to determine the exercise intensity and timing that result in maximal lipid oxidation during prolonged exercise.
In the present study, we investigated how differences in exercise intensity affect the blood hormone response and energy substrate oxidation during exercise performed in the morning and evening.
The study subjects were nine healthy young men who did not exercise regularly (Table 1). Before participation, all subjects received a full explanation outlining the present study and its safety, and provided their written consent to participate. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and was approved by the ethics committees of Waseda University.
Measurement of V[O.sub.2max]
All subjects underwent an incremental exercise test to calculate their V[O.sub.2max]. Measurements were randomly performed twice in each subject between 9:00 am to 10:00 am and 5:00 pm to 6:00 pm. The trials were conducted at least 1 week apart. The exercise test used a treadmill (MAT-2700, Fukuda Denshi) and the Bruce protocol, in which the incline and the speed are increased every 3 min. The amount of expired gas during the exercise test was analyzed with an expired gas analyzer (Aero Monitor AE300s, Minato Medical Science), with mean values for every 30 s calculated by using the breath-bybreath method. V[O.sub.2max] was defined when at least two of the following three conditions were met: 1) plateaued oxygen intake, 2) respiratory exchange ratio (RER) of 1.1 or higher, and 3) heart rate (HR) at 90% of the age-specific maximum predicted HR (220 - age) (Pillard et al., 2010).
Determination of [Fat.sub.max] and MFO [Fat.sub.max] is defined as the exercise intensity with the MFO. MFO is defined as the maximum amount of lipid oxidation observed during the incremental exercise test. Lipid oxidation rates were calculated through indirect calorime-try from gas exchange measurements by using Frayn's equations. For each participant, the rates were calculated after smoothing the curve plotting lipid oxidation as a function of exercise intensity. A digital filter (40 s) was used to smooth the breath-by-breath fluctuations with a width that depended on the amount of noise in the data (Takagi et al., 2014). MFO and [Fat.sub.max] were determined for each participant in the morning and evening.
The subjects were instructed to abstain from strenuous exercise and from consuming alcohol or caffeine from 2 days before the experiment. They were also instructed to eat a provided meal 3 h before the start of the experiment, and were prohibited from consuming food or drink other than those provided. The meal was equivalent to 2430 kJ, and 34.4% of the energy was derived from fat, 59.9% from carbohydrate, and 5.7% from protein. This experiment consisted of four different exercise conditions--a morning and an evening trial at 60%V[O.sub.2max] and [Fat.sub.max] --and all subjects performed all four trials. The four trials were randomized cross-comparison tests and had an at least 1 week interval between them. Exercise was started at 9:00 am and 5:00 pm for the morning and evening trials, respectively, at exercise intensities of 60%V[O.sub.2max] and [Fat.sub.max].
The exercise duration for the 60%V[O.sub.2max] trial was 1 h. On the other hand, the duration of the [Fat.sub.max] (morning: 101.33 [+ or -] 4.71 min, evening: 96.85 [+ or -] 5.91 min) trial was determined for each subject such that the amount of...