Om modified Bergstrom biopsy needle and manually-applied suction. Muscle tissue was immediately blotted, snap-frozen in liquid nitrogen, and stored at 280uC until analysis. Forty-eight hours following the muscle biopsy, participants returned to the lab for a VO2peak incremental ramp test to exhaustion on a Monark Ergomedic 874E stationary ergometer (Vansbro, Sweden). The VO2peak ramp protocol consists of a five minute loadless warm-up followed by a step increase to 70 W for one minute and subsequent increases in work rate of 21 W?min21 to volitional exhaustion or the failure of the participant to maintain a cadence of 60 RPM. Gas exchange and heart rate were measured with a metabolic cart (Moxus AEI Technologies, Pittsburgh, PA) calibrated before each test using a two-point gas calibration (known gas and atmospheric air) and a 3L syringe for volume calibration. Relative VO2peak, absolute VO2peak and HRpeak were selected as the highest value of continuous 30 s averages for each measure during the protocol. Peak O2 pulse was calculated as absolute VO2peak divided by HRpeak from the incremental ramp protocol. Twenty-four hours following the incremental ramp test, participants completed a 500 kcal time to completion trial at a self-selected cadence as quickly as possible against a load expected to elicit 50 VO2peak at 60 RPM [17]. Total external workInterval Training in Overweight/Obese Men(expressed in kcal) was calculated by the cycle ergometer based on load (Title Loaded From File kiloponds) and cycle speed (rpm). Participants were given no temporal, verbal, or physiological feedback and were only aware of how many calories had been expended. Exercise duration was Title Loaded From File recorded for each test.Proteins were visualized by chemiluminescence detection according to the manufacturer’s instructions (Millipore, Billerica, MA). Blots were imaged using the FluorChem Cell Biosciences imaging system and quantified using AlphaView technology. Equal protein loading for all Western blots were confirmed using GAPDH.Training ProtocolTraining was performed three times per week for three weeks with the number of intervals completed per training session increasing each week, starting at 8 intervals per session for Week 1, 9 per session in Week 2, and 10 per session in Week 3. Each training session consisted of a five minute loadless warm-up, followed by repeated 60 s intervals at either 100 (HI) or 70 (LO) of baseline testing maximal aerobic power while maintaining a cadence of 80 RPM. Each interval was followed by 60 s of loadless cycling at 80 RPM and all training sessions ended with a five minute loadless cool down period. To assess perceived affect (i.e., valence feelings of pleasure ?displeasure), the Feeling Scale (FS) was also administered during each participant’s first training session of each week of training. This single-item, validated 11-point Likert bipolar FS ranged from 25 (Very Bad) to +5 (Very Good).Citrate Synthase and b-Hydroxyacyl-CoA Dehydrogenase ActivityAn additional portion of muscle (,20 mg) was homogenized by hand for 30 s in glass hand homogenizers on ice and used to determine maximal citrate synthase (CS) and b-hydroxyacyl-CoA dehydrogenase (bHAD) activity. Total CS activity was measured spectrophotometrically at 37uC using the colormetric agent DTNB at 412 nm. bHAD activity was determined at 37uC by determining the rate of disappearance of NADH at 340 nm.Blood AnalysisFasted blood glucose was determined via a hexokinase reaction assay performed at the K.Om modified Bergstrom biopsy needle and manually-applied suction. Muscle tissue was immediately blotted, snap-frozen in liquid nitrogen, and stored at 280uC until analysis. Forty-eight hours following the muscle biopsy, participants returned to the lab for a VO2peak incremental ramp test to exhaustion on a Monark Ergomedic 874E stationary ergometer (Vansbro, Sweden). The VO2peak ramp protocol consists of a five minute loadless warm-up followed by a step increase to 70 W for one minute and subsequent increases in work rate of 21 W?min21 to volitional exhaustion or the failure of the participant to maintain a cadence of 60 RPM. Gas exchange and heart rate were measured with a metabolic cart (Moxus AEI Technologies, Pittsburgh, PA) calibrated before each test using a two-point gas calibration (known gas and atmospheric air) and a 3L syringe for volume calibration. Relative VO2peak, absolute VO2peak and HRpeak were selected as the highest value of continuous 30 s averages for each measure during the protocol. Peak O2 pulse was calculated as absolute VO2peak divided by HRpeak from the incremental ramp protocol. Twenty-four hours following the incremental ramp test, participants completed a 500 kcal time to completion trial at a self-selected cadence as quickly as possible against a load expected to elicit 50 VO2peak at 60 RPM [17]. Total external workInterval Training in Overweight/Obese Men(expressed in kcal) was calculated by the cycle ergometer based on load (kiloponds) and cycle speed (rpm). Participants were given no temporal, verbal, or physiological feedback and were only aware of how many calories had been expended. Exercise duration was recorded for each test.Proteins were visualized by chemiluminescence detection according to the manufacturer’s instructions (Millipore, Billerica, MA). Blots were imaged using the FluorChem Cell Biosciences imaging system and quantified using AlphaView technology. Equal protein loading for all Western blots were confirmed using GAPDH.Training ProtocolTraining was performed three times per week for three weeks with the number of intervals completed per training session increasing each week, starting at 8 intervals per session for Week 1, 9 per session in Week 2, and 10 per session in Week 3. Each training session consisted of a five minute loadless warm-up, followed by repeated 60 s intervals at either 100 (HI) or 70 (LO) of baseline testing maximal aerobic power while maintaining a cadence of 80 RPM. Each interval was followed by 60 s of loadless cycling at 80 RPM and all training sessions ended with a five minute loadless cool down period. To assess perceived affect (i.e., valence feelings of pleasure ?displeasure), the Feeling Scale (FS) was also administered during each participant’s first training session of each week of training. This single-item, validated 11-point Likert bipolar FS ranged from 25 (Very Bad) to +5 (Very Good).Citrate Synthase and b-Hydroxyacyl-CoA Dehydrogenase ActivityAn additional portion of muscle (,20 mg) was homogenized by hand for 30 s in glass hand homogenizers on ice and used to determine maximal citrate synthase (CS) and b-hydroxyacyl-CoA dehydrogenase (bHAD) activity. Total CS activity was measured spectrophotometrically at 37uC using the colormetric agent DTNB at 412 nm. bHAD activity was determined at 37uC by determining the rate of disappearance of NADH at 340 nm.Blood AnalysisFasted blood glucose was determined via a hexokinase reaction assay performed at the K.