Cryotherapy is widely used in sports and rehabilitation to aid recovery and injury management. The purpose was to examine if a low temperature computer controlled continuous knee cooling protocol (10°C) for one hour and a moderate continuous knee cooling protocol (18°C) for one hour affected neuromuscular activity and functional performance tests. We used a randomized controlled study design. Twenty healthy male subjects (age = 24 ± 3 years) were included and randomized into 2 groups (10°C and 18°C). On day one, participants performed a maximal voluntary contraction of the quadriceps (MVC), single leg hop for distance (SLHD), and crossover hop for time (COHT) with both legs before and after cooling of their right leg. At day two, the same tests were performed with both legs before and after cooling of the left leg. Participants exposed to the 10°C-protocol showed a significant decrease in SLHD and COHT performance. For the 18°C-group, no significant changes in SLHD and COHT outcomes were noted. In both groups, EMG frequency during MVC decreased, but no significant increases were found in EMG amplitude. Continuous knee cooling at 18°C for one hour does not affect functional hop performance, though adaptations at the muscle level (EMG frequency decrease) can be observed. Applying a similar cooling protocol with 10°C results in a significant decrease in functional hop performance and EMG frequency. EMG amplitude remained unaffected. This infers that changes at muscle level due to local temperature manipulations may not always be detrimental to functional performance.