Consider an average email campaign that generated 40% open rate and 5% click rate. This means that 60% of the audience probably saw the subject line and did not open the email and another 35% opened the email but did not find any call to action compelling enough to click. All this information is available to you in real time.
Here is the opportunity to send your second best subject line to everyone that did not open. If this subject line generated 25% opens then you have just boosted your overall opens to 55% of the total audience. That's not all; your clicks went up from 5% of your audience to 8% of the total audience.
The most important trick here is timing, i.e., how long do you wait before you send the second subject line. We generally recommend waiting 3-6 days after sending your original mailing or as soon as your open rates and response rates begin to flatten. Sending the second subject line too soon could generate spam complaints and drive your opt-out rates higher than normal.
Source: Bluestreak's "BlueNotes" newsletter.