To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.