The purpose of this study was to investigate the separate effects of cooling during the acute (within 60 min post-exercise) or subacute (24-168 h post-exercise) phase on skeletal muscle after exercise. Twenty-eight male subjects [mean (SD) 23.8 (1.8) years] were randomly assigned to the control (COTG, n=10), cold-water immersion (CWIG, n=9), and double-cold-water immersion groups (DCWIG, n=9). The cold-water immersion (15 min) was administered to the subjects' legs after calf-raise exercise (CWIG: after recording initial post-exercise measures, DCWIG: after recording initial and 24 h post-exercise measures). Magnetic resonance T2-weighted images were obtained to calculate the T2 relaxation time (T2) of the triceps surae muscle before, immediately after, and at the following times post-exercise: 20, 40, and 60 min, and 24, 48, 96 and 168 h. In addition, the ankle joint range of motion, serum creatine kinase and lactate dehydrogenase, and muscle soreness level were investigated before and after exercise. In all groups, significant T2 elevations in the gastrocnemius muscle appeared from immediately after to 60 min after exercise (P<0.05). Thereafter, COTG showed significantly re-elevated T2 levels in the gastrocnemius at 96-168 h post-exercise (P<0.05), while CWIG and DCWIG showed significantly smaller T2 values than the COTG at 96 h post-exercise (P<0.05). In addition, COTG showed larger increases in serum enzymes at 96 h post-exercise (not significant) and significantly greater muscle soreness levels at 48 h post-exercise (P<0.05) than the cooling groups. The results of this study may suggest that cooling has no dramatic effect, but some minor effects on reducing exercise-induced muscle edema in the subacute phase and relieving the extent of the damaged muscle cells.