Abstract: Kang, J, Mangine, GT, Ratamess, NA, Faigenbaum, AD, and Hoffman, JR. Acute effect of intensity fluctuation on energy output and substrate utilization. J Strength Cond Res 28(8): 2136–2144, 2014—Exercise routines in which intensity fluctuates, such as Spinning and Treading, are gaining in popularity in fitness industry. However, literature on how this dynamic protocol may affect the exercise metabolism is lacking. The present investigation was undertaken to examine the effect of intensity fluctuation and its magnitude on oxygen uptake and substrate utilization during exercise and recovery. Fifteen men and 15 women were randomly assigned into 1 of the 3 groups consisting of 10 participants of equal gender. Each group performed one of the three 30-minute exercise protocols that yielded the same total power output: (a) cycling at a constant power output of 75 W (P1), (b) cycling with power output alternating between 50 and 100 W every 5 minutes (P2), and (c) cycling with power output alternating between 25 and 125 W every 5 minutes (P3). Each exercise session was followed by a 25-minute recovery. Oxygen uptake (V[Combining Dot Above]O2), carbon dioxide production (V[Combining Dot Above]CO2), and respiratory exchanged ratio were measured at rest and during exercise and recovery. Rates of carbohydrate (COX) and fat oxidation (FOX) were calculated based on V[Combining Dot Above]O2 and V[Combining Dot Above]CO2 using the stoichiometric equations. V[Combining Dot Above]O2 in ml·kg−1·min−1 did not differ across the 3 protocols during exercise, but was higher (p ≤ 0.05) in P2 (4.92 ± 0.51) or P3 (4.94 ± 0.24) than P1 (4.17 ± 0.19) during recovery. COX in mg·kg−1·min−1 was higher (p ≤ 0.05) in P3 (17.68 ± 1.30) than in P1 (12.22 ± 1.55) or P2 (12.06 ± 1.47) during exercise and higher in P3 (4.17 ± 0.45) than in P1 (2.60 ± 0.36) during recovery. FOX in mg·kg−1·min−1 was lower (p ≤ 0.05) in P3 (2.61 ± 0.47) than in P1 (4.30 ± 0.60) or P2 (4.22 ± 0.47) during exercise but remained similar across the 3 protocols during recovery. These data indicate that intensity fluctuation of sufficient magnitude can alter exercise metabolism independent of the total power output or overall intensity. The 2 variable intensity protocols used in the study (i.e., P2 and P3) are equally effective in augmenting postexercise V[Combining Dot Above]O2, but the protocol with a greater magnitude of fluctuation also elicits greater COX coupled with reduced FOX during exercise.