Purpose: This study assessed the mitochondrial related signaling responses to a single bout of non-contact, modified football (touch rugby), played as small-sided games (SSG), or cycling (CYC) exercise in sedentary, obese, middle-aged men. Method: In a randomized, cross-over design, nine middle-aged, sedentary, obese men completed two, 40-min exercise conditions (CYC and SSG) separated by a 21-d recovery period. Heart rate (HR) and Ratings of Perceived Exertion were collected during each bout. Needle biopsy from the m. vastus lateralis were collected at rest, 30 and 240min post-exercise for analysis of protein content and phosphorylation (PGC-1a, SIRT1, p53, p53Ser15, AMPK, AMPKThr172, CAMKII, CAMKIIThr286, p38MAPK and p38MAPKThr180/Tyr182) and mRNA expression (PGC-1a, p53, NRF1, NRF2, Tfam and cytochrome-c). Results: A main effect of time effect for both conditions was evident for HR, RPE and blood lactate (P<0.05), with no condition by time interaction (P>0.05). Both conditions increased PGC1-a protein and mRNA expression at 240min (P<0.05). AMPKThr172 increased 30min post CYC (P<0.05), with no change in SSG (P>0.05). CYC increased p53 protein content at 240min to a greater extent than SSG (P<0.05). mRNA expression of NRF2 decreased in both conditions (P<0.05). No condition x time interactions were evident for mRNA expression of Tfam, NRF1, cytochrome-c and p53. Conclusions: The similar PGC-1a response between intensity-matched conditions suggests both conditions are of similar benefit for stimulating mitochondrial biogenesis. Differences between conditions regarding fluctuation in exercise intensity and type of muscle contraction may explain the increase of p53 and AMPK within CYC and not SSG (non-contact, modified football).