The confidential phone service went live last April. In its first 11 months, 90 anonymous calls from NHS Scotland staff raised concerns about health service practices and 69 calls related to personnel or contractual issues. A further 222 calls were taken from health workers from other parts of the UK.
Health Improvement Scotland is looking at six of the cases for further investigation.
Health Secretary Alex Neil said it was an "additional safe place" where staff could raise concerns in confidence.
Mr Neil said: "I have always been clear that there is no place for gagging staff in NHS Scotland. This builds on the changes that I have recently announced to remove confidentiality clauses from standard settlement agreements for NHS staff."
The telephone whistleblower service is expected to cost about £90,000 over two years.
The NHS Confidential Alert Line can be contacted on 0800 008 6112.