Cellular skeletal muscle lipid metabolism is of paramount importance for metabolic health, specifically through its connection to branched-chain amino acids (BCAA) metabolism and through its modulation by exercise. In this study, we aimed at better understanding intramyocellular lipids (IMCL) and their related key proteins in response to physical activity and BCAA deprivation. By means of confocal microscopy, we examined IMCL and the lipid droplet coating proteins PLIN2 and PLIN5 in human twin pairs discordant for physical activity. Additionally, in order to study IMCLs, PLINs and their association to peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in cytosolic and nuclear pools, we mimicked exercise-induced contractions in C2C12 myotubes by electrical pulse stimulation (EPS), with or without BCAA deprivation. The life-long physically active twins displayed an increased IMCL signal in type I fibers when compared to their inactive twin pair. Moreover, the inactive twins showed a decreased association between PLIN2 and IMCL. Similarly, in the C2C12 cell line, PLIN2 dissociated from IMCL when myotubes were deprived of BCAA, especially when contracting. In addition, in myotubes, EPS led to an increase in nuclear PLIN5 signal and its associations with IMCL and PGC-1α. This study demonstrates how physical activity and BCAA availability affects IMCL and their associated proteins, providing further and novel evidence for the link between the BCAA, energy and lipid metabolisms.